Subtilisin DY variants having decreased adsorption and increased hydrolysis

ABSTRACT

The present invention relates to Subtilisin DY variants having a modified amino acid sequence of wild-type Subtilisin DY amino acid sequence, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region, a fifth loop region and a sixth loop region; wherein the modified amino acid sequence comprises different amino acids than that occurring in wild-type Subtilisin DY (i.e., substitution) at specifically identified positions in one or more of the loop regions whereby the Subtilisin DY variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type Subtilisin DY. The present invention also relates to DNA sequences encoding such Subtilisin DY variants. The present invention also relates to compositions comprising such Subtilisin DY variants for cleaning a variety of surfaces.

TECHNICAL FIELD

The present invention relates to novel enzyme variants useful in a variety of cleaning compositions, and DNA sequences encoding such enzyme variants.

BACKGROUND

Enzymes make up the largest class of naturally occurring proteins. Each class of enzyme generally catalyzes (accelerates a reaction without being consumed) a different kind of chemical reaction. One class of enzymes known as proteases, are known for their ability to hydrolyze (break down a compound into two or more simpler compounds with the uptake of the H and OH parts of a water molecule on either side of the chemical bond cleaved) other proteins. This ability to hydrolyze proteins has been taken advantage of by incorporating naturally occurring and protein engineered proteases as an additive to laundry detergent preparations. Many stains on clothes are proteinaceous and wide-specificity proteases can substantially improve removal of such stains.

Unfortunately, the efficacy level of these proteins in their natural, bacterial environment, frequently does not translate into the relatively unnatural wash environment. Specifically, protease characteristics such as thermal stability, pH stability, oxidative stability and substrate specificity are not necessarily optimized for utilization outside the natural environment of the enzyme.

The amino acid sequence of the protease determines the characteristics of the protease. A change of the amino acid sequence of the protease may alter the properties of the enzyme to varying degrees, or may even inactivate the enzyme, depending upon the location, nature and/or magnitude of the change in the amino acid sequence. Several approaches have been taken to alter the wild-type amino acid sequence of proteases in an attempt to improve their properties, with the goal of increasing the efficacy of the protease in the wash environment. These approaches include altering the amino acid sequence to enhance thermal stability and to improve oxidation stability under quite diverse conditions.

Despite the variety of approaches described in the art, there is a continuing need for new effective variants of proteases useful for cleaning a variety of surfaces.

OBJECTS OF THE PRESENT INVENTION

It is an object of the present invention to provide Subtilisin DY enzyme variants having improved hydrolysis versus the wild-type of the enzyme.

It is also an object of the present invention to provide cleaning compositions comprising these subtilisin enzyme variants.

SUMMARY

The present invention relates to Subtilisin DY variants having a modified amino acid sequence of wild-type Subtilisin DY amino acid sequence, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region and a fifth loop region; wherein the modified amino acid sequence comprises different amino acids than that occurring in wild-type Subtilisin DY (i.e., substitution) at specifically identified positions in one or more of the loop regions whereby the Subtilisin DY variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type Subtilisin DY. The present invention also relates to DNA sequences encoding such Subtilisin DY variants. The present invention also relates to compositions comprising such Subtilisin DY variants for cleaning a variety of surfaces.

DESCRIPTION

I. Subtilisin DY Variants

This invention pertains to subtilisin enzymes, in particular Subtilisin DY, that have been modified by mutating the various nucleotide sequences that code for the enzyme, thereby modifying the amino acid sequence of the enzyme. The modified subtilisin enzymes (hereinafter, “Subtilisin DY variants”) of the present invention have decreased adsorption to and increased hydrolysis of an insoluble substrate as compared to the wild-type subtilisin. The present invention also pertains to DNA sequences encoding for such Subtilisin DY variants.

The subtilisin enzymes of this invention belong to a class of enzymes known as proteases. A protease is a catalyst for the cleavage of peptide bonds. One type of protease is a serine protease. A serine protease is distinguished by the fact that there is an essential serine residue at the active site.

The observation that an enzyme's rate of hydrolysis of soluble substrates increases with enzyme concentration is well documented. It would therefore seem plausible that for surface bound substrates, such as is encountered in many cleaning applications, the rate of hydrolysis would increase with increasing surface concentration. This has been shown to be the case. (Brode, P. F. III and D. S. Rauch, Langmur, “Subtilisin BPN′: Activity on an Immobilized Substrate”, Vol. 8, pp. 1325-1329 (1992)). In fact, a linear dependence of rate upon surface concentration was found for insoluble substrates when the surface concentration of the enzyme was varied. (Rubingh, D. N. and M. D. Bauer, “Catalysis of Hydrolysis by Proteases at the Protein-Solution Interface,” in Polymer Solutions, Blends And Interfaces, Ed. by I. Noda and D. N. Rubingh, Elsevier, p. 464 (1992)). Surprisingly, when seeking to apply this principle in the search for variant proteases which give better cleaning performance, we did not find that enzymes which adsorb more give better performance. In fact, we surprisingly determined the opposite to be the case: decreased adsorption by an enzyme to a substrate resulted in increased hydrolysis of the substrate (i.e., better cleaning performance).

While not wishing to be bound by theory, it is believed that improved performance, when comparing one variant to another, is a result of the fact that enzymes which adsorb less are also less tightly bound and therefore more highly mobile on the surface from which the insoluble protein substrate is to be removed. At comparable enzyme solution concentrations, this increased mobility is sufficient to outweigh any advantage that is conferred by delivering a higher concentration of enzyme to the surface.

The mutations described herein are designed to change (i.e., decrease) the adsorption of the enzyme to surface-bound soils. In Subtilisin DY, certain amino acids form exterior loops on the enzyme molecule. For purposes of discussion, these loops shall be referred to as first, second, third, fourth and fifth loop regions. Specifically, positions 5865 form the first loop region; positions 94-106 form the second loop region; positions 125-132 form the third loop region; positions 153-166 form the fourth loop region; positions 186-190 form the fifth loop region; and positions 199-219 form the sixth loop region (position numbering analagous to positions in the amino acid sequence for wild-type subtilisin Subtilisin DY (SEQ ID NO:1)).

It is believed that these loop regions play a significant role in the adsorption of the enzyme molecule to a surface-bound peptide, and specific mutations in one or more of these loop regions will have a significant effect on this adsorption. While not wishing to be bound by theory, it is believed that the loop regions are important to the adsorption of the Subtilisin DY molecule for at least two reasons. First, the amino acids which comprise the loop regions can make close contacts with any surfaces to which the molecule is exposed. Second, the proximity of the loop regions to the active-site and binding pocket of the Subtilisin DY molecule gives them a role in the catalytically productive adsorption of the enzyme to surface-bound substrates (peptides/protein soils).

As used herein, “variant” means an enzyme having an amino acid sequence which differs from that of wild-type.

As used herein, “mutant Subtilisin DY DNA” means a DNA sequence coding for a Subtilisin DY variant.

As used herein, “wild-type Subtilisin DY” refers to an enzyme represented by SEQ ID NO:1. The amino acid sequence for Subtilisin DY is further described by Nedkov, P., Oberthur, W. and Braunitzer, G., Biol. Chem., Vol. 366, pp. 421430 (1985), incorporated herein by reference.

As used herein, the term “Subtilisin DY wild-type amino acid sequence” encompasses SEQ ID NO:1 as well as SEQ ID NO:1 having modifications to the amino acid sequence other than at any of positions 58, 59, 60, 61, 62, 64, 65, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 125, 126, 127, 128, 129, 130, 131, 132, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 186, 187, 188, 189, 190, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218 or 219.

As used herein, “more hydrophilic amino acid” refers to any other amino acid having greater hydrophilicity than a subject amino acid with reference to the hydrophilicity table below. The following hydrophilicity table (Table 1) lists amino acids in descending order of increasing hydrophilicity (see Hopp, T. P., and Woods, K. R., “Prediction of Protein Antigenic Determinants from Amino Acid Sequences”, Proceedings of the National Academy of Science USA, Vol. 78, pp. 3824-3828, 1981, incorporated herein by reference).

TABLE 1 Amino Acid Hydrophilicity Value Trp −3.4 Phe −2.5 Tyr −2.3 Leu, Ile −1.8 Val −1.5 Met −1.3 Cys −1.0 Ala, His −0.5 Thr −0.4 Pro, Gly −0.0 Gln, Asn 0.2 Ser 0.3 Arg⁺, Lys⁺, Glu⁻, Asp⁻ 3.0

Table 1 also indicates which amino acids carry a charge (this characteristic being based on a pH of from about 8-9). The positively charged amino acids are Arg and Lys, the negatively charged amino acids are Glu and Asp, and the remaining amino acids are neutral. In a preferred embodiment of the present invention, the substituting amino acid is either neutral or negatively charged, more preferably negatively charged (i.e., Glu or Asp).

Therefore, for example, the statement “substitute Gln with an equally or more hydrophilic amino acid which is neutral or has a negative charge” means Gln would be substituted with Asn (which is equally hydrophilic to Gln), or Ser, Glu or Asp (which are more hydrophilic than Gln); each of which are neutral or have a negative charge, and have a greater hydrophilicity value as compared to Gln. Likewise, the statement “substitute Pro with a more hydrophilic amino acid which is neutral or has a negative charge” means Pro would be substituted with Gln, Asn, Ser, Glu or Asp.

In one embodiment of the present invention, the Subtilisin DY variant has a modified amino acid sequence of Subtilisin DY wild-type amino acid sequence, wherein the wild-type amino acid sequence comprises a substitution at one or more positions in one or more of the first loop region, the second loop region, the third loop region, the fourth loop region, the fifth loop region or the sixth loop region; whereby the Subtilisin DY variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type Subtilisin DY.

In a preferred embodiment of the present invention, the substituting amino acid for one or more of the positions in one or more of the loop regions is, with reference to Table 1, neutral or negatively charged and equally or more hydrophylic, preferably more hydrophylic, than the amino acid at the subject position in the wild-type amino acid sequence.

A. Substitutions in the First Loop Region

When a substitution occurs in the first loop region, the substitution occurs at one or more of positions 58, 59, 60, 61, 62, 64 or 65.

When a substitution occurs at position 58, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.

When a substitution occurs at position 59, the substituting amino acid is Glu.

When a substitution occurs at position 60, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs at position 61, the substituting amino acid is Asp, Gln, Glu or Ser.

When a substitution occurs at position 62, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs at position 65, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.

B. Substitutions in the Second Loop Region

When a substitution occurs in the second loop region, the substitution occurs at one or more of positions 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106.

When a substitution occurs at position 94, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr.

When a substitution occurs at position 95, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val.

When a substitution occurs at position 96, the substituting amino acid is Asp, Gln, Glu or Ser.

When a substitution occurs at position 97, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 98, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 99, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs at position 100, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 101, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs at position 102, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.

When a substitution occurs at position 103, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val.

When a substitution occurs at position 104, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 105, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr.

When a substitution occurs at position 106, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val.

C. Substitutions in the Third Loop Region

When a substitution occurs in the third loop region, the substitution occurs at one or more of positions 125, 126, 127, 128, 129, 130, 131 or 132.

When a substitution occurs at position 125, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val.

When a substitution occurs at position 126, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs at position 128, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser.

When a substitution occurs at position 129, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 130, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs at position 131, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 132, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.

D. Substitutions in the Fourth Loop Region When a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 or 166.

When a substitution occurs at position 153, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs at position 154, the substituting amino acid is Asp, Gln, Glu or Ser.

When a substitution occurs at position 155, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs at position 157, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 158, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 159, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs at position 160, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 161, the substituting amino acid is Asn, Asp, Glu or Ser.

When a substitution occurs at position 162, the substituting amino acid is Asp, Gln, Glu or Ser.

When a substitution occurs at position 163, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.

When a substitution occurs at position 164, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val.

When a substitution occurs at position 165, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs at position 166, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val.

E. Substitutions in the Fifth Loop Region

When a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 186, 187, 188, 189 or 190.

When a substitution occurs at position 186, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr.

When a substitution occurs at position 187, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 188, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val.

When a substitution occurs at position 189, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 190, the substituting amino acid is Asp or Glu.

F. Substitutions in the Sixth Loop Region

When a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218 or 219.

When a substitution occurs at position 199, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr.

When a substitution occurs at position 200, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser.

When a substitution occurs at position 201, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs at position 202, the substituting amino acid is Ala, Asn, Asp, Cys, gin, Glu, Gly, His, Met, Pro, Ser or Thr.

When a substitution occurs at position 203, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 204, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser, or Thr.

When a substitution occurs at position 205, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val.

When a substitution occurs at position 206, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 207, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.

When a substitution occurs at position 208, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr or Val.

When a substitution occurs at position 209, the substituting amino acid is Asn, Asp, gin, Glu, Gly, or Ser.

When a substitution occurs at position 210, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 21 1, the substituting amino acid is Asp, Gln, Glu or Ser.

When a substitution occurs at position 212, the substituting amino acid is Asn, Asp, gin, Glu, Gly, Pro or Ser.

When a substitution occurs at position 213, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val.

When a substitution occurs at position 214, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.

When a substitution occurs at position 215, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 216, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val.

When a substitution occurs at position 217, the substituting amino acid is Asp, gin, Glu or Ser.

When a substitution occurs at position 218, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs at position 219, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.

G. Preparation of Enzyme Variants

EXAMPLE 1 Mutant Subtilisin DY DNA

A phagemid (“DYP”) containing the wild type Subtilisin DY gene is constructed. The 2.8 Kbp Pvu II restriction enzyme fragment of plasmid pUC119, (Vieira, J. and Messing, J., “Production of Single-Stranded Plasmid DNA”, 153 Methods in Enzymology 3-11 (1989)) is cloned into the Pvu II site of plasmid pUB110 (Bacillus Genetic Stock Center, Columbus, Ohio 1E9). The pUC119-pUB110 hybrid plasmid is named pJMA601. Into the BamHI restriction site of pJMA601 is cloned the polymerase chain reaction—amplified subtilisin DY gene from chromosomal DNA giving phagemid DYP. Phagemid DYP is transformed into Escherichia coli Ung⁻ strain CJ236 and a single stranded uracil-containing DNA template is produced using the VCSM13 helper phage (Kunkel, T. A., J. D. Roberts and R. A. Zakour, “Rapid and efficient site-specific mutagenesis without phenotypic selection”, Methods in Enzymology, VOl. 154, pp. 367-382, (1987); as modified by Yuckenberg, P. D., F. Witney, J. Geisselsoder and J. McClary, “Site-directed in vitro mutagenesis using uracil-containing DNA and phagemid vectors”, Directed Mutagenesis-A Pratical Approach, ed. M. J. McPherson, pp. 2748, (1991); both of which are incorporated herein by reference). A single primer site-directed mutagenesis modification of the method of Zoller and Smith (Zoller, M. J., and M. Smith, “Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA”, Nucleic Acids Research, Vol. 10, pp. 6487-6500, (1982), incorporated herein by reference) is used to produce all mutants (basically as presented by Yuckenberg, et al., 1991, above). Oligonucleotides are made using an Applied Biosystem Inc. 380B DNA synthesizer. Mutagenesis reaction products are transformed into Escherichia coli strain MM294 (American Type Culture Collection E. coli. 33625). All mutants are confirmed by DNA sequencing and the isolated DNA is transformed into the Bacillus subtilis expression strain BG2036 (Yang, M. Y., E. Ferrari and D. J. Henner, (1984), “Cloning of the Neutral Protease Gene of Bacillus subtilis and the Use of the Cloned Gene to Create an In Vitro-derived Deletion Mutation”, Journal of Bacterilogy, Vol. 160, pp. 15-21). For some of the loop mutants a modified DYP with a frameshift-stop codon mutation in the corresponding loop is used to produce the uracil template. Oligonucleotides are designed to restore the proper reading frame and to encode for random substitutions at positions 58, 59, 60, 61, 62, 64, 65, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 125, 126, 127, 128, 129, 130, 131, 132, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 186, 187, 188, 189, 190, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218 or 219 (equimolar and/or variable mixtures of all four nucleotides for all three bases at these codons). Mutations that correct for the frameshift-stop and produce a functional enzyme are identified by their ability to digest casein. The random substitutions are determined by DNA sequencing.

EXAMPLE 2 Fermentation

The Bacillus subtilis cells (BG2036) containing a subtilisin mutant of interest are grown to mid-log phase in a one liter culture of LB-glucose broth and inoculated into a Biostat ED fermenter (B. Braun Biotech, Inc., Allentown, Pa.) in a total volume of 10 liters. The fermentation media contains Yeast Extract, starch, antifoam, buffers and trace minerals (see Fermentation: a Practical Approach, Ed. B. McNeil and L. M. Harvey, 1990). The broth is kept at a constant pH of 7.0 during the fermentation run. Chloramphenical is added for antibiotic selection of mutagenized plasmid. The cells are grown overnight at 37° C. to an A₆₀₀ of about 60 and harvested.

EXAMPLE 3 Purification

The fermentation broth is taken through the following steps to obtain pure enzyme. The broth is cleared of Bacillus subtilis cells by centrifugation, and clarified by removing fine particulates with a 100K cutoff membrane. This is followed by concentration on a 10K cutoff membrane, and flow dialysis to reduce the ionic strength and adjust the pH to 5.5 using 0.025M MES buffer (2-(N-morpholino)ethanesulfonic acid). The enzyme is further purified by loading it onto either a cation exchange chromatography column or an affinity adsorption chromatography column and eluting it from the column with a NaCl or a propylene glycol gradient (see Scopes, R. K., Protein Purification Principles and Practice, Springer-Verlag, New York (1984), incorporated herein by reference).

The pNA assay (DelMar, E. G., C. Largman, J. W. Brodrick and M. C. Geokas, Anal. Biochem., Vol. 99, pp. 316-320, (1979), incorporated herein by reference) is used to determine the active enzyme concentration for fractions collected during gradient elution. This assay measures the rate at which p-nitroaniline is released as the enzyme hydrolyzes the soluble synthetic substrate, succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (sMPF-pNA). The rate of production of yellow color from the hydrolysis reaction is measured at 410 nm on a spectrophotometer and is proportional to the active enzyme concentration. In addition, absorbance measurements at 280 nm are used to determine the total protein concentration. The active enzyme/total-protein ratio gives the enzyme purity, and is used to identify fractions to be pooled for the stock solution.

To avoid autolysis of the enzyme during storage, an equal weight of propylene glycol is added to the pooled fractions obtained from the chromatography column. Upon completion of the purification procedure the purity of the stock enzyme solution is checked with SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the absolute enzyme concentration is determined via an active site titration method using trypsin inhibitor type II-T: turkey egg white purchased from Sigma Chemical Company (St. Louis, Mo.). The measured conversion factors will show which changes made in the enzyme molecule at the various positions result in the enzyme variant having increased activity over the wild-type, against the soluble substrate pNA.

In preparation for use, the enzyme stock solution is eluted through a Sephadex-G25 (Pharmacia, Piscataway, N.J.) size exclusion column to remove the propylene glycol and exchange the buffer. The MES buffer in the enzyme stock solution is exchanged for 0.1 M Tris buffer (Tris(hydroxymethyl-aminomethane) containing 0.01M CaCl₂ and pH adjusted to 8.6 with HCl. All experiments are carried out at pH 8.6 in Tris buffer thermostated at 25° C.

H. Characterization of Enzyme Variants

EXAMPLE 4 Model Surface Preparation

Aminopropyl controlled pore glass (CPG) purchased from CPG Inc. (Fairfield, N.J.) is used as a support for covalently attaching the sAAPF-pNA substrate purchased from Bachem, Inc. (Torrence, Calif.). The reaction is carried out in dimethyl sulfoxide and (1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide hydrochloride) (EDC) is used as a coupling agent. Upon completion (monitored by pNA assay), the excess solvent is removed, and the CPG:sMPF-pNA is rinsed with dimethyl sulfoxide (DMSO) and doubly-distilled water. This is followed by oven drying with a N₂ purge at about 70° C. The reaction scheme and preparation of the immobilized substrate are conducted as described by Brode, P. F. III, and D. S. Rauch, “Subtilisin BPN′: Activity on an Immobilized Substrate,” Langmur, Vol. 8, p. 1325-1329, (1992), incorporated herein by reference.

The CPG surface will have 62,000±7,000 pNA molecules/μm². The surface area will remain unchanged from the value of 50.0 m²/g reported by CPG Inc. for the CPG as received. This suggests that the procedure used to add sAAPF-pNA to CPG does not damage the porous structure (mean diameter is 486 Å).

EXAMPLE 5 Surface Hydrolysis Assay

Using CPG:sAAPF-pNA, adsorption of an enzyme variant and hydrolysis of a CPG-bound peptide can be measured in a single experiment. A small volume of enzyme variant stock solution is added to a flask containing Tris buffer and CPG:sAAPF-pNA which has been degassed. The flask is shaken on a wrist-action shaker for a period of 90 minutes during which the shaker is stopped at various time intervals (for example, every 2 minutes during the early stages of adsorption hydrolysis—e.g., the first 20 minutes—and every 10 minutes towards the end of the experiment). The CPG:sAAPF-pNA is allowed to settle and the solution is sampled. Both the experimental procedure and the calculation of the adsorption and hydrolysis are conducted as described by Brode et al., 1992, above.

All enzymes are monitored for stability against autolysis and should show no appreciable autolytic loss over the time course of this experiment. Therefore, enzyme adsorption can be determined by measuring solution depletion. The difference between the initial enzyme variant concentration and the concentration measured at each individual time point gives the amount of enzyme variant adsorbed. The amount of pNA hydrolyzed from the surface is measured by taking an absorbance reading on an aliquot of the sample at 410 nm. The total amount of pNA hydrolyzed is calculated by adding the amount sampled and the amount remaining in the flask. This value is corrected by subtracting the amount of pNA that is hydrolyzed by Tris buffer at pH 8.6 when no enzyme is present. This base-hydrolysis ranges from 7-29% of the total hydrolysis depending on the efficiency of the enzyme.

EXAMPLE 6 Soluble Substrate Kinetic Analysis

The rates of hydrolysis of the soluble substrate sAAPF-pNA are monitored by measuring the adsorbance increase as a function of time at 410 nm on a DU-70 spectrophotometer. The enzyme concentration is held constant and is prepared to be in the range of 6-10 nanomolar while the substrate concentration is varied from 90-700 μM sAAPF-pNA for each kinetic determination. An adsorbance data point is taken each second over a period of 900 seconds and the data are transferred to a Lotus™ spreadsheet (Lotus Development Corporation, Cambridge, Mass.). Analysis for kinetic parameters is conducted by the standard Lineweaver Burk analysis in which the data in the initial part of the run (generally the first minute) are fit to a linear regression curve to give v_(O). The v_(O) and s_(O) data are plotted in the standard inverse fashion to give K_(M) and k_(cat).

I. Example Subtilisin DY Variants

Subtilisin DY variants of the present invention which have decreased adsorption to and increased hydrolysis of surface bound substrates are exemplified in Tables 2-36, below. In describing the specific mutations, the original amino acid occurring in wild-type is given first, the position number second, and the substituted amino acid third.

TABLE 2 Loop 1 - Single Mutation Variants Thr58Asn Thr58Asp Thr58Gln Thr58Glu Thr58Gly Thr58Pro Thr58Ser Asp59Glu Gly60Asn Gly60Asp Gly60Gln Gly60Glu Gly60Pro Gly60Ser Asn61Asp Asn61Gln Asn61Glu Asn61Ser Gly62Asn Gly62Asp Gly62Gln Gly62Glu Gly62Pro Gly62Ser Gly64Asn Gly64Asp Gly64Gln Gly64Glu Gly64Pro Gly64Ser Thr65Asn Thr65Asp Thr65Gln Thr65Glu Thr65Gly Thr65Pro Thr65Ser

TABLE 3 Loop 1 - Double Mutation Variants Thr58Gln + Asn61Glu Thr58Asn + Asn61Asp Asp59Glu + Gly64Pro Thr58Glu + Gly62Pro Asp59Glu + Gly60Ser Gly60Asn + Gly64Asn Asn61Asp + Thr65Gln Thr58Asp + Asn61Ser Thr58Asp + Thr65Pro Gly64Glu + Thr65Gly Gly64Asp + Thr65Asn Thr58Glu + Gly62Gln Gly60Asn + Asn61Glu Thr58Asn + Gly64Ser Thr58Pro + Asp59Glu Gly64Glu + Thr65Gln Thr58Pro + Gly62Ser Thr58Gly + Gly64Asn Asn61Gln + Gly64Gln Thr58Gly + Asn61Asp Gly60Pro + Asn61Asp Gly62Asn + Gly64Pro Gly62Asp + Thr65Asn Thr58Asn + Gly62Asn Thr58Pro + Gly62Asn Gly64Glu + Thr65Ser Gly60Pro + Gly62Glu Gly60Glu + Thr65Gln Gly60Pro + Gly62Ser Thr58Asp + Asn61Gln Gly60Asn + Asn61Asp Thr58Ser + Gly62Glu Gly60Glu + Gly64Ser Gly60Pro + Asn61Glu Thr58Gln + Asn61Ser Gly60Asp + Thr65Pro Asn61Asp + Thr65Ser Asn61Ser + Gly64Asp

TABLE 4 Loop 1 - Triple Mutation Variants Thr58Asn + Gly60Gln + Gly64Pro Thr58Gly + Asn61Ser + Thr65Ser Asp59Glu + Gly60Pro + Thr65Ser Asn61Glu + Gly62Pro + Gly64Asn Thr58Asp + Gly60Asn + Thr65Gln Thr58Ser + Gly62Ser + Thr65Gly Thr58Pro + Asp59Glu + Gly62Pro Gly60Glu + Asn61Gln + Thr65Gln Gly60Glu + Gly64Asn + Thr65Pro Gly60Asn + Gly62Asp + Thr65Ser Thr58Asn + Asn61Glu + Thr65Gln Thr58Asn + Asp59Glu + Gly62Pro Gly60Gln + Gly62Gln + Gly64Glu Asn61Gln + Gly62Pro + Thr65Ser Asn61Gln + Gly62Asn + Thr65Gln Asn61Ser + Gly62Gln + Thr65Gly Gly60Ser + Gly64Gln + Thr65Gly Thr58Pro + Gly60Pro + Gly64Gln Thr58Asn + Gly60Asn + Thr65Pro Asp59Glu + Gly62Gln + Thr65Asn Thr58Ser + Gly60Gln + Gly62Asp Thr58Asn + Gly64Ser + Thr65Asn Gly60Pro + Gly62Asp + Thr65Gln Asn61Glu + Gly64Ser + Thr65Gly Thr58Pro + Asn61Asp + Gly64Gln Asp59Glu + Asn61Ser + Gly62Ser Thr58Asn + Gly60Asp + Thr65Asn Asp59Glu + Asn61Gln + Gly62Asn Thr58Asn + Gly60Asp + Asn61Asp Thr58Asn + Gly60Glu + Asn61Glu

TABLE 5 Loop 1 - Quadruple Mutation Variants Thr58Asn + Gly60Glu + Gly64Pro + Thr65Gln Asp59Glu + Gly60Ser + Gly64Pro + Thr65Pro Thr58Ser + Gly60Asn + Asn61Gln + Thr65Pro Asp59Glu + Gly60Ser + Gly64Ser + Thr65Gly Asp59Glu + Gly60Asn + Asn61Ser + Gly62Ser Asp59Glu + Gly60Asn + Asn61Ser + Gly64Asn Thr58Gln + Asn61Gln + Gly62Glu + Gly64Asn Thr58Asn + Asp59Glu + Gly60Gln + Thr65Pro Thr58Gly + Gly60Gln + Gly62Pro + Gly64Glu Gly60Asp + Asn61Asp + Gly62Pro + Gly64Gln Thr58Pro + Gly60Glu + Asn61Asp + Gly64Asn Thr58Asp + Asp59Glu + Gly62Asn + Thr65Gln Thr58Asp + Asp59Glu + Gly60Ser + Gly64Asn Thr58Glu + Asp59Glu + Gly62Asn + Thr65Ser Thr58Asp + Asp59Glu + Gly60Pro + Asn61Ser Thr58Asn + Gly60Gln + Asn61Glu + Gly62Asp Asp59Glu + Gly60Glu + Asn61Glu + Gly62Ser Thr58Gln + Asp59Glu + Gly60Glu + Asn61Glu Thr58Pro + Asp59Glu + Gly60Glu + Asn61Glu Thr58Gln + Asp59Glu + Gly60Asp + Asn61Asp Thr58Pro + Gly60Glu + Asn61Asp + Gly62Glu Gly60Glu + Asn61Glu + Gly62Glu + Thr65Gly Gly60Asp + Asn61Asp + Gly62Asp + Thr65Gln Thr58Asp + Asp59Glu + Gly60Glu + Thr65Ser Asp59Glu + Asn61Asp + Gly62Asp + Thr65Gln Asp59Glu + Asn61Asp + Gly62Glu + Thr65Gly Asp59Glu + Gly60Asp + Gly62Asp + Thr65Gln Thr58Ser + Asp59Glu + Gly60Asp + Gly62Asp

TABLE 6 Loop 2 - Single Mutation Variants Val 94Ala Val94Asn Val94Asp Val94Cys Val94Gln Val94Glu Val94Gly Val94His Val94Met Val94Pro Val94Ser Val94Thr Leu95Ala Leu95Asn Leu95Asp Leu95Cys Leu95Gln Leu95Glu Leu95Gly Leu95His Leu95Ile Leu95Met Leu95Pro Leu95Ser Leu95Thr Leu95Val Asn96Asp Asn96Gln Asn96Glu Asn96Ser Ser97Asp Ser97Glu Ser98Asp Ser98Glu Gly99Asn Gly99Asp Gly99Gln Gly99Glu Gly99Pro Gly99Ser Ser100Asp Ser100Glu Gly101Asn Gly101Asp Gly101Gln Gly101Glu Gly101Pro Gly101Ser Thr102Asn Thr102Asp Thr102Gln Thr102Glu Thr102Gly Thr102Pro Thr102Ser Tyr103Ala Tyr103Asn Tyr103Asp Tyr103Cys Tyr103Gln Tyr103Glu Tyr103Gly Tyr103His Tyr103Ile Tyr103Leu Tyr103Met Tyr103Pro Tyr103Ser Tyr103Thr Tyr103Val Ser104Asp Ser104Glu Ala105Asn Ala105Asp Ala105Gln Ala105Glu Ala105Gly Ala105His Ala105Pro Ala105Ser Ala105Thr Ile106Ala Ile106Asn Ile106Asp Ile106Cys Ile106Gln Ile106Glu Ile106Gly Ile106His Ile106Leu Ile106Met Ile106Pro Ile106Ser Ile106Thr Ile106Val

TABLE 7 Loop 2 - Double Mutation Variants Val94Gln + Ser100Glu Asn96Ser + Gly99Gln Val94Asp + Thr102Gln Val94His + Ala105Pro Val94Pro + Ser97Glu Asn96Ser + Ile106Asp Ser97Glu + Gly101Ser Leu95Gly + Thr102Glu Ser100Glu + Ile106Thr Asn96Ser + Gly99Ser Ala105Ser + Ile106Pro Asn96Ser + Gly101Ser Thr102Asn + Ile106Gln Val94His + Asn96Gln Leu95Cys + Ala105Asp Leu95Gly + Asn96Asp Leu95Cys + Asn96Asp Thr102Ser + Ile106Cys Asn96Gln + Thr102Gln Gly101Ser + Tyr103Gly Asn96Ser + Ile106Thr Ser98Asp + Gly99Gln Leu95Ser + Ser100Glu Leu95Met + Thr102Gly Leu95His + Thr102Glu Asn96Asp + Tyr103His Tyr103Gln + Ile106Asp Ser104Asp + Ile106Gln Thr102Asn + Tyr103Asp Val94Asn + Ser97Glu Val94Gly + Gly101Ser Leu95Gln + Ile106Asp Asn96Ser + Ser104Asp Tyr103Glu + Ile106His Thr102Ser + Tyr103Glu Val94Cys + Ser104Asp Leu95Gln + Thr102Asp Asn96Glu + Gly101Ser Leu95Glu + Ala105Ser Val94Met + Leu95Pro Val94Thr + Gly101Gln Tyr103Gly + Ile106His Thr102Asn + Ile106Gly Ser104Asp + Ile106Thr Gly99Ser + Ser100Asp Val94Glu + Gly99Asn Gly99Gln + Gly101Ser Val94Thr + Asn96Asp Ser97Glu + Ile106Leu Thr102Asp + Tyr103Met Ser97Asp + Ala105His Asn96Gln + Ser100Asp Gly101Asn + Ile106Gln Asn96Glu + Tyr103Ser Tyr103Thr + Ile106Pro Ser100Asp + Tyr103Ile Gly101Glu + Tyr103Leu Ser100Asp + Thr102Asn Val94His + Leu95Thr Ser104Asp + Ala105Asn

TABLE 8 Loop 2 - Triple Mutation Variants Val94Gln + Leu95Thr + Ser100Glu Ser98Asp + Ala105Ser + Ile106Asn Val94Gly + Gly99Pro + Ile106Val Val94Ser + Leu95Gln + Ser104Glu Val94His + Tyr103Glu + Ile106Gly Val94Pro + Asn96Gln + Ile106Cys Val94Thr + Ser98Asp + Ile106Cys Val94Met + Ser100Asp + Gly101Ser Val94Ala + Ser97Asp + Gly99Gln Leu95Gly + Gly99Pro + Ala105Gly Val94Cys + Ser100Asp + Tyr103Ala Gly101Asn + Thr102Asp + Ile106Ala Gly99Ser + Ser104Glu + Ile106Val Asn96Gln + Ser100Glu + Tyr103Thr Leu95Met + Thr102Pro + Ile106Leu Asn96Glu + Thr102Ser + Ala105His Ser100Glu + Thr102Asn + Tyr103Val Ser98Glu + Gly99Gln + Ile106Met Asn96Asp + Gly99Ser + Ala105Thr Gly101Asp + Thr102Asn + Tyr103Thr Val94Ala + Gly99Gln + Tyr103His Thr102Gly + Tyr103Glu + Ala105Gly Ser97Glu + Gly101Ser + Thr102Gln Val94Pro + Gly99Ser + Gly101Asp Ser97Asp + Gly99Ser + Ile106Gly Val94Cys + Leu95Val + Gly101Gln Ser100Asp + Gly101Asn + Tyr103Gln Thr102Asp + Tyr103Leu + Ile106Val Val94Cys + Ser104Glu + Ala105Asn Val94Ser + Thr102Asn + Ala105Thr Ser98Glu + Gly99Asn + Tyr103Val Leu95Val + Gly101Gln + Ala105Asp Ser97Glu + Gly99Pro + Thr102Ser Val94Ser + Gly101Glu + Ile106Ala Gly99Asn + Gly101Asp + Thr102Glu Gly99Asp + Ser100Glu + Thr102Asn Val94Thr + Gly99Glu + Ser100Asp Gly99Asp + Ser100Asp + Tyr103Pro Gly99Glu + Ser100Glu + Gly101Gln Asn96Glu + Ser97Glu + Ala105Asn Asn96Glu + Ser97Asp + Gly101Pro Leu95Met + Asn96Asp + Ser97Glu Gly99Ser + Ser104Asp + Ala105Glu Ser97Asp + Ser98Glu + Gly101Ser Asn96Asp + Ser98Asp + Gly99Glu Asn96Glu + Ser97Glu + Gly99Glu Leu95Asp + Ser100Glu + Ile106Leu Asn96Asp + Ser100Glu + Gly101Glu Ser104Asp + Ala105Thr + Ile106Glu Asn96Glu + Ser98Asp + Tyr103Ala Leu95Asn + Ser98Asp + Ser100Glu Leu95Gly + Ser98Glu + Ser100Glu Ser98Asp + Ser100Glu + Ala105Ser Ser98Glu + Ser100Asp + Thr102Asn Val94Asn + Ser97Glu + Gly99Glu Asn96Glu + Ser100Glu + Thr102Pro Val94Asp + Gly101Asp + Thr102Pro Gly101Glu + Thr102Asp + Ser104Asp Ser98Glu + Ser100Asp + Gly101Asp Leu95Glu + Gly101Asp + Ile106Glu

TABLE 9 Loop 2 - Quadruple Mutation Variants Gly99Asn + Gly101Gln + Thr102Glu + Tyr103Val Val94Asn + Gly101Asp + Tyr103Ile + Ala105Thr Val94Asn + Gly99Ser + Ser100Glu + Thr102Ser Leu95Cys + Ser98Asp + Gly101Pro + Ile106Ala Leu95His + Gly101Ser + Tyr103Ile + Ala105Ser Val94Ser + Leu95Val + Gly99Pro + Ser100Glu Gly99Ser + Ser100Glu + Tyr103Ile + Ala105Ser Val94Ala + Ser97Glu + Thr102Ser + Ala105Pro Val94Thr + Tyr103Gly + Ser104Asp + Ala105Gln Val94Ala + Leu95Pro + Ser100Glu + Ile106Ser Leu95Asn + Ser98Asp + Tyr103Gly + Ala105Pro Asn96Ser + Gly101Glu + Tyr103Leu + Ala105Pro Leu95Met + Ser98Glu + Gly99Asn + Tyr103Thr Val94Ala + Leu95Ala + Ser97Asp + Thr102Asn Gly99Gln + Ser100Glu + Thr102Ser + Ile106Leu Asn96Glu + Gly99Gln + Ala105Pro + Ile106Thr Val94Gly + Gly101Glu + Tyr103Gln + Ile106His Val94Thr + Ser97Asp + Gly101Gln + Ala105Thr Val94Gly + Leu95Val + Asn96Gln + Ser104Glu Leu95Gln + Gly99Glu + Ser100Glu + Thr102Pro Leu95Met + Ser100Asp + Gly101Asp + Tyr103Ser Leu95Ile + Thr102Pro + Ser104Asp + Ala105Asp Ser97Asp + Ser98Asp + Tyr103Met + Ala105Pro Leu95Ser + Ser97Asp + Ser98Asp + Thr102Ser Ser97Glu + Ser98Glu + Ala105Pro + Ile106Asn Ser97Glu + Ser98Glu + Gly101Ser + Ile106Cys Ser97Asp + Ser98Asp + Gly101Pro + Thr102Pro Leu95Glu + Ser100Glu + Gly101Asp + Thr102Ser Val94Met + Leu95Glu + Asn96Glu + Gly101Asp Leu95His + Gly99Glu + Ser100Glu + Gly101Glu Asn96Glu + Gly99Glu + Gly101Ser + Ile106Asn Leu95Val + Asn96Asp + Ser98Glu + Tyr103Gln Asn96Asp + Ser98Glu + Gly99Ser + Thr102Asn Asn96Glu + Ser98Glu + Gly101Ser + Thr102Pro Leu95Val + Asn96Glu + Ser98Asp + Ile106Ala Val94Gly + Asn96Glu + Ser98Asp + Tyr103Thr Ser98Glu + Gly99Gln + Ser100Asp + Gly101Pro Ser98Glu + Gly99Gln + Ser100Asp + Thr102Gln Asn96Glu + Ser100Glu + Tyr103Gln + Ile106Pro Asn96Asp + Gly99Asn + Ser100Glu + Tyr103Ile Leu95Glu + Asn96Gln + Ser98Glu + Gly99Glu Ser97Asp + Ser98Glu + Ser100Asp + Ile106Val Ser97Asp + Ser98Asp + Ser100Glu + Thr102Ser Ser97Asp + Ser98Asp + Ser100Glu + Gly101Gln Leu95Thr + Ser97Asp + Ser98Glu + Ser100Asp Val94Met + Asn96Glu + Gly99Asp + Gly101Asp Ser98Asp + Ser100Glu + Gly101Glu + Ala105Ser Val94Cys + Ser98Asp + Ser100Asp + Gly101Asp Val94Glu + Ser100Glu + Gly101Glu + Tyr103Ile Leu95Glu + Ser97Asp + Ser98Asp + Ala105Ser Leu95Ala + Asn96Asp + Gly101Asp + Thr102Asp Val94Cys + Gly99Asp + Gly101Asp + Ile106Pro Leu95Ser + Ser100Glu + Gly101Gln + Thr102Glu Leu95Met + Ser100Asp + Thr102Glu + Ile106Gln Asn96Ser + Gly101Asp + Tyr103Asn + Ile106Asp Leu95Cys + Gly101Glu + Ala105Pro + Ile100Glu Val94Asp + Gly99Glu + Ser100Asp + Ala105Ser Ser97Glu + Ser98Glu + Gly101Asp + Tyr103Ser Ser97Asp + Ser100Glu + Thr102Gln + Tyr103Thr Ser97Glu + Ser100Asp + Tyr103Ser + Ala105Gly

TABLE 10 Loop 3 - Single Mutation Variants Leu125Ala Leu125Asn Leu125Asp Leu125Cys Leu125Gln Leu125Glu Leu125Gly Leu125His Leu125Ile Leu125Met Leu125Pro Leu125Ser Leu125Thr Leu125Val Gly126Asn Gly126Asp Gly126Gln Gly126Glu Gly126Pro Gly126Ser Gly127Asn Gly127Asp Gly127Gln Gly127Glu Gly127Pro Gly127Ser Pro128Asn Pro128Asp Pro128Gln Pro128Glu Pro128Gly Pro128Ser Ser129Asp Ser129Glu Gly130Asn Gly130Asp Gly130Gln Gly130Glu Gly130Pro Gly130Ser Ser131Asp Ser131Glu Thr132Asn Thr132Asp Thr132Gln Thr132Glu Thr132Gly Thr132Pro Thr132Ser

TABLE 11 Loop 3 - Double Mutation Variants Leu125Gln + Ser129Glu Leu125Pro + Gly126Ser Gly127Gln + Ser131Asp Gly130Ser + Thr132Gly Leu125Met + Gly130Asp Leu125Pro + Pro128Asp Ser129Asp + Gly130Asn Leu125Asp + Pro128Asn Gly127Ser + Ser131Glu Leu125Ile + Gly130Ser Leu125Thr + Thr132Asp Leu125Thr + Thr132Asn Leu125Pro + Gly127Glu Leu125Glu + Thr132Asn Pro128Asn + Thr132Pro Leu125Thr + Gly130Asp Gly127Pro + Thr132Ser Gly127Asn + Thr132Asn Gly126Gln + Ser131Asp Leu125Val + Thr132Gly Gly126Gln + Gly130Ser Ser129Asp + Thr132Gly Gly126Asp + Gly130Pro Leu125Cys + Pro128Gln Gly127Gln + Thr132Gly Leu125His + Gly126Asp Leu125Val + Ser129Glu Gly126Ser + Pro128Asp Leu125Val + Gly126Glu Gly127Glu + Pro128Ser Leu125Asn + Thr132Pro Leu125Met + Gly126Asp Leu125Ser + Ser129Glu Leu1253er + Gly126Asn Leu125Val + Thr132Gln Leu125Val + Gly130Asp Ser129Glu + Thr132Ser Pro128Gly + Ser129Asp Gly126Pro + Thr132Asp Gly126Asp + Thr132Ser Pro128Asp + Thr132Gln Gly126Pro + Ser129Asp Gly130Ser + Thr132Glu Gly127Glu + Pro128Gln Gly126Asn + Gly130Pro Pro128Asn + Ser129Glu Gly130Asp + Thr132Gly Gly126Gln + Ser131Glu Gly127Glu + Gly130Ser Gly126Asn + Ser129Glu Pro128Asp + Gly130Asn Pro128Asp + Thr132Gly Gly126Gln + Pro128Asp Ser131Glu + Thr132Gly Gly130Glu + Thr132Asn Leu125Val + Pro128Gly Leu125Ile + Gly130Pro Pro128Asn + 3er131Asp Gly130Asn + Thr132Asp Gly127Ser + Pro128Glu

TABLE 12 Loop 3 - Triple Mutation Variants Gly127Asp + Gly130Gln + Thr132Ser Pro128Asp + Gly130Prb + Thr132Ser Gly127Asn + Ser131Asp + Thr132Pro Leu125Ser + Ser131Asp + Thr132Pro Leu125Met + Pro128Glu + Thr132Pro Leu125Asp + Gly126Ser + Thr132Gln Gly127Asn + Pro128Glu + Gly130Asn Gly127Ser + Pro128Gln + Ser129Glu Gly126Pro + Gly127Pro + Ser131Glu Leu125Ile + Gly127Asp + Thr132Gln Gly126Asn + Gly130Ser + Thr132Pro Gly126Pro + Gly127Gln + Thr132Asp Gly126Asp + Gly130Asn + Thr132Gln Leu125Ser + Pro128Gln + Thr132Asn Gly126Pro + Gly127Pro + Pro128Glu Leu125Met + Gly126Glu + Gly130Gln Leu125Met + Ser129Asp + Thr132Gln Leu125His + Ser129Asp + Gly130Ser Gly127Pro + Ser129Glu + Gly130Ser Pro128Gln + Gly130Asn + Thr132Gln Gly126Asn + Gly127Pro + Gly130Gln Leu125Pro + Gly126Ser + Gly127Gln Leu125His + Gly127Ser + Ser131Glu Leu125His + Gly127Asn + Thr132Glu Gly126Glu + Gly130Pro + Thr132Gln Leu125His + Gly127Gln + Gly130Glu Gly127Pro + Pro128Gly + Thr132Asn Leu125Gly + Pro128Gly + Gly130Asp Gly126Asn + Pro128Asn + Ser129Glu Gly126Pro + Gly130Ser + Thr132Gly Leu125Ser + Gly126Pro + Ser131Glu Leu125Val + Gly126Glu + Thr132Gly Pro128Asn + Ser131Asp + Thr132Pro Leu125Gly + Pro128Asn + Thr132Pro Gly127Glu + Pro128Ser + Thr132Pro Gly127Gln + Ser129Glu + Thr132Gln Leu125Ile + Gly127Pro + Gly130Asp Gly126Ser + Gly127Asp + Gly130Asn Pro128Glu + Gly130Pro + Thr132Gln Leu125Val + Gly126Asp + Gly127Asp Pro128Asn + Gly130Asp + Ser131Asp Gly130Glu + Ser131Asp + Thr132Gln Gly126Pro + Gly130Glu + Ser131Asp Gly127Glu + Pro128Glu + Thr132Ser Gly130Gln + Ser131Glu + Thr132Asp Leu125Gln + Ser131Asp + Thr132Asp Gly130Ser + Ser131Glu + Thr132Glu Gly130Pro + Ser131Asp + Thr132Asp Gly127Pro + Ser131Glu + Thr132Asp Leu125Asn + Ser131Asp + Thr132Asp Pro128Gln + Ser131Asp + Thr132Glu Pro128Gly + Ser131Asp + Thr132Asp Gly126Gln + Ser129Asp + Gly130Asp Leu125Ile + Ser129Asp + Gly130Glu Gly127Glu + Pro128Asp + Ser129Glu Ser129Asp + Gly130Asp + Ser131Asp Ser129Glu + Gly130Asp + Ser131Glu Ser129Glu + Gly130Glu + Ser131Asp Gly126Glu + Gly127Glu + Pro128Glu Gly130Glu + Ser131Asp + Thr132Asp

TABLE 13 Loop 3 - Quadruple Mutation Variants Gly126Ser + Pro128Glu + Gly130Ser + Thr132Ser Leu125Cys + Gly127Gln + Ser129Glu + Thr132Pro Leu125Gln + Gly126Pro + Pro128Ser + Ser129Glu Leu125Thr + Ser129Glu + Gly130Ser + Thr132Pro Gly127Gln + Pro128Gln + Ser129Glu + Gly130Pro Gly127Asn + Pro128Asn + Ser131Glu + Thr132Gly Leu125Met + Gly127Glu + Gly130Asn + Thr132Gly Leu125Met + Gly127Asn + Pro128Asn + Ser129Glu Leu125Met + Gly126Ser + Gly127Gln + Ser131Asp Leu125Asn + Gly126Asp + Gly127Gln + Thr132Ser Leu125Gln + Gly127Gln + Gly130Glu + Thr132Pro Leu125Ala + Gly130Asn + Ser131Asp + Thr132Pro Leu125Met + Gly126Pro + Gly127Ser + Ser131Asp Leu125Cys + Gly126Pro + Ser129Glu + Thr132Asn Leu125Gly + Pro128Gly + Ser131Asp + Thr132Gln Leu125Met + Gly130Asn + Ser131Asp + Thr132Gln Leu125Val + Gly126Pro + Gly130Gln + Thr132Asp Gly126Asn + Gly127Pro + Pro128Gln + Ser129Glu Leu125Gln + Gly126Glu + Gly127Glu + Gly130Asn Leu125Val + Gly126Asp + Gly127Glu + Gly130Pro Leu125Asp + Gly126Glu + Gly127Pro + Thr132Pro Leu125Glu + Gly126Glu + Gly127Asn + Thr132Ser Leu125Gln + Gly127Pro + Gly130Glu + Ser131Asp Leu125Cys + Gly130Glu + Ser131Asp + Thr132Gln Gly126Gln + Gly127Glu + Pro128Glu + Gly130Asn Gly126Ser + Gly127Asp + Pro128Asp + Gly130Ser Leu125Thr + Gly127Ser + Ser131Glu + Thr132Asp Pro128Asn + Gly130Asn + Ser131Asp + Thr132Glu Leu125Met + Gly126Ser + Ser129Asp + Gly130Asp Gly126Gln + Pro128Gln + Ser129Glu + Gly130Glu Gly127Asn + Pro128Asn + Ser129Glu + Gly130Glu Gly127Ser + Ser129Glu + Gly130Asp + Thr132Gln Gly127Asp + Pro128Glu + Ser129Asp + Gly130Asn Gly127Asp + Pro128Glu + Ser129Asp + Thr132Ser Leu125Gln + Ser129Glu + Gly130Asp + Ser131Asp Leu125Cys + Ser129Glu + Gly130Asp + Ser131Glu Gly127Gln + Pro128Glu + Ser129Glu + Gly130Asp Leu125Met + Gly126Asp + Gly127Glu + Pro128Glu Gly127Glu + Pro128Gln + Ser129Glu + Thr132Ser Leu125Pro + Gly127Asp + Pro128Ser + Ser129Asp Gly126Pro + Gly127Glu + Pro128Gly + Ser129Asp Leu125Pro + Gly127Asp + Ser129Glu + Gly130Glu Gly127Asp + Pro128Ser + Ser129Glu + Gly130Asp Gly127Asp + Ser129Glu + Gly130Glu + Thr132Pro Leu125Ile + Gly127Glu + Ser129Asp + Gly130Asp Gly126Glu + Gly127Asp + Ser129Glu + Gly130Pro Gly126Asp + Gly127Glu + Ser129Asp + Thr132Pro Leu125Met + Gly126Asp + Gly127Asp + Ser129Asp Gly126Ser + Ser129Asp + Ser131Glu + Thr132Gln Gly127Gln + Pro128Asn + Ser129Asp + Ser131Asp Pro128Gln + Ser129Glu + Ser131Asp + Thr132Ser Gly126Pro + Ser129Asp + Ser131Glu + Thr132Ser Leu125Ala + Ser129Glu + Gly130Pro + Ser131Asp Gly127Gln + Ser129Glu + Gly130Ser + Ser131Asp Gly127Ser + Ser129Glu + Ser131Glu + Thr132Gln Gly127Gln + Pro128Gln + Ser129Glu + Ser131Asp Leu125Ala + Gly126Ser + Ser129Asp + Ser131Glu Gly126Pro + Pro128Gln + Ser129Asp + Ser131Asp Gly126Ser + Pro128Ser + Ser129Asp + Ser131Glu Gly126Glu + Pro128Asp + Ser129Asp + Thr132Asn

TABLE 14 Loop 4 - Single Mutation Variants Gly153Asn Gly153Asp Gly153Gln Gly153Glu Gly153Pro Gly153Ser Asn154Asp Asn154Gln Asn154Glu Asn154Ser Ser155Asp Ser155Glu Gly156Asn Gly156Asp Gly156Gln Gly156Glu Gly156Pro Gly156Ser Ser157Asp Ser157Glu Ser158Asp Ser158Glu Gly159Asn Gly159Asp Gly159Gln Gly159Glu Gly159Pro Gly159Ser Ser160Asp Ser160Glu Gln161Asn Gln161Asp Gln161Glu Gln161Ser Asn162Asp Asn162Gln Asn162Glu Asn162Ser Thr163Asn Thr163Asp Thr163Gln Thr163Glu Thr163Gly Thr163Pro Thr163Ser Ile164Ala Ile164Asn Ile164Asp Ile164Cys Ile164Gln Ile164Glu Ile164Gly Ile164His Ile164Leu Ile164Met Ile164Pro Ile164Ser Ile164Thr Ile164Val Gly165Asn Gly165Asp Gly165Gln Gly165Glu Gly165Pro Gly165Ser Tyr166Ala Tyr166Asn Tyr166Asp Tyr166Cys Tyr166Gln Tyr166Glu Tyr166Gly Tyr166His Tyr166Ile Tyr166Leu Tyr166Met Tyr166Pro Tyr166Ser Tyr166Thr Tyr166Val

TABLE 15 Loop 4 - Double Mutation Variants Asn154Ser + Ser155Glu Gly165Glu + Tyr166Cys Ser158Asp + Gly165Pro Ser157Asp + Thr163Asn Gly156Pro + Thr163Ser Asn154Glu + Ile164Cys Gly153Asn + Thr163Asn Thr163Glu + Ile164Gly Gly156Gln + Ile164Thr Ser157Asp + Ile164His Ser158Glu + Tyr166His Gly153Pro + Tyr166Asp Gly153Pro + Gly165Gln Ser157Glu + Gly159Asn Asn154Ser + Gly159Pro Gly156Glu + Asn162Gln Gln161Asp + Ile164Met Ser157Asp + Thr163Ser Ser157Glu + Tyr166Ile Gln161Glu + Ile164His Ser157Glu + Asn162Gln Gly159Pro + Tyr166Asp Asn162Asp + Ile164Cys Gln161Ser + Asn162Asp Asn154Asp + Asn162Gln Ser158Glu + Gly165Asn Ile164Met + Tyr166Val Gln161Asn + Gly165Pro Ser155Asp + Gly159Pro Ser160Asp + Asn162Ser Gly156Glu + Ile164Cys Thr163Asp + Gly165Pro Gly153Asn + Ser155Glu Ser158Asp + Thr163Gly Ser160Asp + Ile164Met Gly159Asp + Thr163Gln Ser155Glu + Tyr166Cys Gln161Ser + Tyr166Gly Ser160Asp + Ile164Ala Ser157Asp + Gln161Ser Ser160Glu + Asn162Gln Gly153Asn + Gly156Gln Ser157Asp + Ile164Val Asn162Gln + Thr163Glu Ser155Asp + Gln161Ser Gly159Glu + Gly165Gln Ser160Glu + Ile164His Gly153Ser + Ser160Asp Gly159Gln + Gly165Glu Gly153Glu + Gly159Gln Gly156Glu + Thr163Gly Gly156Ser + Tyr166Asn Gly153Asp + Thr163Asn Asn154Ser + Ser157Glu Thr163Ser + Ile164Pro Ser155Glu + Asn162Ser Gly156Pro + Ile164Gly Thr163Glu + Gly165Asn Gly153Gln + Ser155Glu IIe164Ser + Tyr166Cys

TABLE 16 Loop 4 - Triple Mutation Variants Gly153Gln + Asn154Ser + Ser155Glu Ser155Asp + Thr163Pro + Tyr166Gln Gly156Gln + Thr163Glu + Ile164Gly Gly159Gln + Gln161Ser + Gly165Ser Asn154Gln + Thr163Asp + Tyr166Gln Asn154Gln + Gly159Pro + Ile164Thr Asn162Gln + Ile164Gln + Gly165Glu Gly153Asp + Gln161Ser + Thr163Pro Gly153Gln + Asn154Gln + Tyr166Ile Asn154Gln + Gln161Asn + Tyr166Pro Gly153Gln + Ser158Glu + Tyr166Ser Ser157Asp + Ile164Val + Tyr166Ala Thr163Gly + Gly165Asp + Tyr166Pro Gln161Glu + Asn162Gln + Tyr166Thr Gly156Pro + Gln161Glu + Thr163Ser Ser158Glu + Gly159Pro + Gln161Asn Ser158Glu + Gln161Asn + Asn162Ser Asn154Glu + Gln161Ser + Gly165Asn Gly153Asn + Asn154Gln + Ser158Asp Ser157Glu + Ile164Val + Tyr166Val Ser155Asp + Gln161Asn + Tyr166Ile Gly153Glu + Gln161Asn + Gly165Ser Gly156Pro + Asn162Asp + Gly165Pro Gly156Asn + Asn162Asp + Ile164Leu Asn154Gln + Thr163Asn + Gly165Ser Ser158Asp + Gly159Asn + Gln161Asn Ser155Glu + Gln161Asn + Tyr166Leu Ser158Glu + Asn162Ser + Tyr166His Asn154Asp + Thr163Gln + Ile164Met Gly156Asp + Ser157Asp + Gly159Ser Gly159Asp + Ser160Glu + Thr163Gln Ser158Asp + Gly159Asp + Tyr166Cys Asn154Glu + Ser155Glu + Gly156Ser Ser155Asp + Gly156Asp + Asn162Ser Ser157Asp + Ser158Glu + Gly159Pro Ser160Asp + Gln161Asp + Ile164Ala Gly159Gln + Ser160Asp + Gln161Asp Gly156Asp + Ser157Asp + Ser158Asp Gly159Asp + Gln161Glu + Gly165Gln Gly153Pro + Gly159Glu + Gln161Glu Ser157Glu + Ser158Asp + Gln161Glu Ser157Glu + Gly159Glu + Gly165Ser Ser157Glu + Gly159Glu + Ser160Glu Ser158Asp + Ser160Asp + Ile164Thr Ser158Asp + Ser160Glu + Tyr166Val Asn154Gln + Ser158Asp + Ser160Asp Ser158Glu + Ser160Asp + Tyr166His Gly153Asp + Asn154Glu + Gly165Glu Ser155Asp + Ser157Asp + Tyr166Ile Ser155Glu + Ser157Asp + Gly159Pro Gly153Asn + Ser157Glu + Thr163Glu Ser155Asp + Ser157Asp + Ser158Glu Ser157Asp + Gly159Asp + Gln161Glu Ser155Asp + Gly156Glu + Ser158Asp Asn154Gln + Ser160Glu + Asn162Asp Ser158Glu + Ser160Glu + Asn162Glu Ser157Glu + Gln161Asp + Thr163Asp Asn154Glu + Ser155Glu + Ser157Glu Ser157Asp + Ser160Glu + Tyr166Gly Gly153Asn + Ser157Glu + Ser160Asp

TABLE 17 Loop 4 - Quadruple Mutation Variants Asn154Ser + Gly159Asp + Ile164Met + Gly165Pro Ser155Glu + Gly156Ser + Asn162Gln + Thr163Asn Gly156Gln + Ser157Asp + Gly159Ser + Asn162Ser Ser155Asp + Gly159Pro + Gln161Ser + Ile164Cys Asn154Gln + Gly156Asp + Thr163Gly + Tyr166Met Asn154Gln + Ser155Asp + Thr163Asn + Gly165Ser Gln161Ser + Asn162Gln + Thr163Gly + Tyr166Gly Ser155Asp + Gly159Pro + Gly165Pro + Tyr166Gln Asn154Ser + Ser155Asp + Thr163Gly + Ile164Val Gly159Asp + Gln161Ser + Thr163Gln + Ile164Thr Gly153Pro + Ser158Asp + Asn162Gln + Ile164Cys Gly153Pro + Gly156Asp + Ile164Ser + Tyr166Leu Gly153Glu + Gly156Pro + Gln161Ser + Tyr166Met Ser155Glu + Thr163Ser + Gly165Pro + Tyr166Leu Ser157Asp + Thr163Gly + Ile164Gln + Tyr166Gly Gly153Ser + Ser155Asp + Thr163Pro + Ile164Pro Asn154Ser + Gly159Gln + Gln161Glu + Tyr166Met Asn154Ser + Ser160Asp + Ile164Gln + Gly165Ser Gly159Asn + Asn162Glu + Gly165Gln + Tyr166Cys Gly156Pro + Gly159Pro + Ile164Gln + Gly165Gln Gly153Ser + Gly156Asn + Asn162Gln + Gly165Asp Ser158Asp + Gly159Glu + Gln161Ser + Ile164Thr Gly153Gln + Asn154Glu + Ser155Glu + Tyr166Gly Gly153Ser + Ser155Asp + Gly156Asp + Ile164Gln Asn154Ser + Ser155Glu + Gly156Asp + Tyr166Val Ser155Glu + Gly156Glu + Thr163Asn + Ile164Leu Ser157Glu + Ser158Asp + Gln161Asn + Ile164Cys Ser160Glu + Gln161Asp + Thr163Gln + Gly165Pro Ser157Asp + Ser158Glu + Gly159Asp + Ile164Cys Asn154Gln + Ser160Glu + Gln161Glu + Asn162Asp Gly153Glu + Gly159Gln + Gln161Asn + Gly165Glu Asn154Ser + Ser158Asp + Gly159Asn + Gln161Glu Asn154Glu + Gly156Asp + Thr163Ser + Tyr166Asn Ser158Glu + Ser160Asp + Gln161Asn + Thr163Pro Asn154Glu + Ser155Asp + Gly156Gln + Thr163Asp Ser158Glu + Gln161Glu + Thr163Asp + Ile164His Ser155Glu + Ser157Asp + Asn162Ser + Gly165Pro Ser155Asp + Ser157Asp + Gly159Pro + Ile164Cys Gly159Glu + Ser160Asp + Asn162Glu + Ile164Asn Ser157Glu + Gln161Asp + Thr163Asp + Gly165Gln Asn154Glu + Ser155Glu + Ser157Asp + Gly159Pro Asn154Gln + Ser160Asp + Gln161Asp + Thr163Asp Ser160Asp + Gln161Asp + Asn162Gln + Thr163Asp Ser155Asp + Thr163Asp + Ile164Gly + Gly165Asp Gly156Ser + Ser158Asp + Thr163Asp + Ile164Leu Ser155Glu + Ser158Glu + Thr163Glu + Ile164Met Gly153Glu + Gly156Pro + Thr163Asp + Tyr166His Gly156Ser + Ser157Asp + Ser160Glu + Asn162Asp Gly153Asn + Ser157Glu + Asn162Asp + Ile164Leu Gly153Glu + Ser155Glu + Ser157Asp + Ile164Met Gly156Pro + Ser157Asp + Gly159Gln + Ser160Glu Gly153Pro + Ser155Glu + Gly156Glu + Gly165Asp Ser155Asp + Ser158Glu + Gln161Asn + Gly165Pro Ser155Asp + Ser158Glu + Gly159Gln + Ile164Gly Gly153Asp + Ser157Glu + Gly159Pro + Thr163Glu Ser155Asp + Gly156Pro + Gln161Ser + Gly165Asp Gly156Glu + Ser157Glu + Ser160Asp + Ile164Ser Asn154Ser + Gly156Asp + Ser158Asp + Ser160Asp Gly153Gln + Ser155Asp + Ser158Asp + Gly159Glu Asn154Asp + Gly156Glu + Ser158Glu + Ile164Ala

TABLE 18 Loop 5 - Single Mutation Variants Ala186Asn Ala186Asp Ala186Gly Ala186Gln Ala186Pro Ala186Ser Ala186Thr Ser187Asp Ser187Glu Phe188Ala Phe188Asn Phe188Asp Phe188Cys Phe188Gln Phe188Glu Phe188Gly Phe188His Phe188Ile Phe188Leu Phe188Met Phe188Pro Phe188Ser Phe188Thr Phe188Tyr Phe188Val Ser189Asp Ser189Glu Ser190Asp Ser190Glu

TABLE 19 Loop 5 - Double Mutation Variants Phe188Met + Ser190Glu Ala186Ser + Ser187Glu Phe188Ser + Ser190Asp Ala186His + Ser187Glu Ser187Asp + Phe188Cys Ala186Ser + Ser190Glu Ala186Pro + Ser190Asp Phe188Ser + Ser190Glu Ala186Gln + Ser187Glu Phe188Asn + Ser190Asp Ala186Glu + Phe188Met Ala186Ser + Phe188Ala Ser187Glu + Phe188Thr Ala186Thr + Phe188Asp Ser187Asp + Phe188Leu Ala186Gly + Phe188Cys Phe188Val + Ser190Glu Ala186Asp + Phe188Pro Ala186Asn + Ser187Asp Phe188Leu + Ser190Glu Ser187Asp + Phe188Asn Ala186Ser + Ser187Asp Phe188Ala + Ser190Glu Ala186Gly + Ser187Glu Ala186Glu + Phe188His Ala186Gly + Phe188Glu Ser187Glu + Phe188Gly Ala186Glu + Phe188Asn Ser187Glu + Phe188His Ala186Ser + Phe188Asp Ala186Gly + Ser187Asp Phe188Ile + Ser190Glu Ala186Thr + Ser190Glu Ala186Gly + Ser190Glu Phe188His + Ser190Asp Phe188Leu + Ser190Asp Ala186Ser + Ser190Asp Ser187Glu + Phe188Ile Ala186Thr + Phe188His Phe188Gln + Ser190Glu Ala186His + Ser187Asp Phe188Tyr + Ser190Glu Ala186Pro + Phe188Thr Ala186Gln + Ser187Asp Ser187Glu + Phe188Ala Ala186Asp + Phe188Ile Ala186Ser + Phe188Pro Ala186Asn + Ser187Glu Phe188His + Ser190Glu Ala186Glu + Phe188Ser Ala186Ser + Phe188Leu Phe188Pro + Ser190Glu Ser187Asp + Phe188Thr Ala186His + Phe188Ala Ala186Asn + Phe188Asp Phe188Ile + Ser190Asp Ala186Thr + Phe188Met Ala186Asp + Phe188His Ser187Asp + Phe188Ala Ala186Pro + Ser190Glu

TABLE 20 Loop 5 - Triple Mutation Variants Ala186His + Phe188Ala + Ser190Asp Ala186Asn + Ser187Asp + Phe188Ala Ala186Gly + Phe188Asn + Ser190Asp Ala186Pro + Phe188Met + Ser190Asp Ala186Gly + Phe188Leu + Ser190Glu Ala186Gln + Ser187Asp + Phe188Ile Ala186Thr + Ser187Glu + Phe188Cys Ala186Ser + Phe188Ala + Ser190Glu Ala186Gln + Ser187Asp + Phe188Ser Ala186Gln + Ser187Glu + Phe188Ile Ala186Ser + Phe188Gly + Ser190Asp Ala186Ser + Ser187Asp + Phe188Met Ala186Gly + Ser187Glu + Phe188Gly Ala186Gln + Phe188Pro + Ser190Asp Ala186Asn + Phe188Ala + Ser190Glu Ala186Ser + Phe188Pro + Ser190Glu Ala186Asn + Ser187Glu + Phe188Pro Ala186Gln + Phe188Gly + Ser190Glu Ala186Asn + Ser187Glu + Phe188Ile Ala186His + Ser187Glu + Phe188Gly Ala186His + Phe188Cys + Ser190Asp Ala186Thr + Ser187Glu + Phe188Val Ala186Gln + Ser187Glu + Phe188Met Ala186His + Phe188Ala + Ser190Glu Ala186Gly + Phe188Met + Ser190Glu Ala186Ser + Ser187Glu + Phe188His Ala186Asn + Phe188Gly + Ser190Glu Ala186Asn + Phe188Pro + Ser190Asp Ala186Asn + Phe188Tyr + Ser190Asp Ala186Gly + Phe188Thr + Ser190Glu Ala186His + Ser187Asp + Phe188Leu Ala186Ser + Phe188Pro + Ser190Asp Ala186Thr + Ser167Glu + Phe188Pro Ala186Pro + Phe188Pro + Ser190Glu Ala186Thr + Phe188Asn + Ser190Asp Ala186Pro + Ser187Glu + Phe188His Ala186Asp + Ser187Glu + Phe188Met Ala186Asp + Ser187Glu + Phe188Gln Ala186Asp + Ser187Glu + Phe188Cys Ala186Glu + Ser187Asp + Phe188Pro Ala186Asp + Ser187Glu + Phe188Tyr Ala186Glu + Ser187Asp + Phe188Thr Ala186Asp + Ser187Asp + Phe188Val Ala186Glu + Ser187Glu + Phe188Asn Ala186Asp + Ser187Asp + Phe188His Ala186Asp + Ser187Glu + Phe188Ala Ala186His + Ser187Asp + Phe188Asp Ala186Gly + Ser187Asp + Phe188Asp Ala186Gln + Ser187Glu + Phe188Glu Ala186Asn + Ser187Asp + Phe188Glu Ala186Pro + Ser187Glu + Phe188Asp Ala186Asp + Ser187Glu + Phe188Asp Ala186Glu + Ser187Glu + Phe188Glu Ala186Asp + Ser187Asp + Phe188Glu Ala186Thr + Phe188Asp + Ser190Glu Ala186Ser + Phe188Glu + Ser190Glu Ala186Thr + Phe188Glu + Ser190Asp Ala186Pro + Phe188Glu + Ser190Glu Ser187Glu + Phe188Glu + Ser190Glu Ser187Asp+ Phe188Asp + Ser190Glu

TABLE 21 Loop 5 - Quadruple Mutation Variants Ala186Gly + Ser187Glu + Phe188Asp + Ser190Glu Ala186Gly + Ser187Asp + Phe188Asp + Ser190Asp Ala186Gln + Ser187Asp + Phe188Asp.+ Ser190Asp Ala186Gln + Ser187Glu + Phe188Asp + Ser190Glu Ala186Ser + Ser187Asp + Phe188Glu + Ser190Glu Ala186Pro + Ser187Glu + Phe188Asp + Ser190Asp Ala186Thr + Ser187Glu + Phe188Asp + Ser190Asp Ala186Thr + Ser187Glu + Phe188Asp + Ser190Glu Ala186His + Ser187Asp + Phe188Glu + Ser190Asp Ala186Asn + Ser187Glu + Phe188Asp + Ser190Asp Ala186Gly + Ser187Glu + Phe188Glu + Ser190Asp Ala186Gln + Ser187Asp + Phe188Glu + Ser190Glu Ala186Gln + Ser187Asp + Phe188Glu + Ser190Asp Ala186Gly + Ser187Glu + Phe188Asp + Ser190Asp Ala186Gln + Ser187Glu + Phe188Gly + Ser190Asp Ala186Asp + Ser187Asp + Phe188Gln + Ser190Asp Ala186Glu + Ser187Glu + Phe188Val + Ser190Asp Ala186Asp + Ser187Glu + Phe188Pro + Ser190Glu Ala186Glu + Ser187Asp + Phe188Met + Ser190Glu Ala186Glu + Ser187Asp + Phe188Ala + Ser190Glu Ala186Asp + Ser187Asp + Phe188His + Ser190Asp Ala186Glu + Ser187Glu + Phe188Met + Ser190Glu Ala186Asp + Ser187Asp + Phe188Ala + Ser190Glu Ala186Glu + Ser187Glu + Phe188Cys + Ser190Asp Ala186Glu + Ser187Glu + Phe188Thr + Ser190Glu Ala186Asp + Ser187Asp + Phe188His + Ser190Glu Ala186Asp + Ser187Asp + Phe188Gln + Ser190Glu Ala186Asp + Ser187Asp + Phe188Tyr + Ser190Asp Ala186Glu + Ser187Asp + Phe188Ser + Ser190Asp Ala186Glu + Ser187Asp + Phe188Gly + Ser190Asp Ala186Asp + Ser187Asp + Phe188Asn + Ser190Asp Ala186Glu + Ser187Glu + Phe188Cys + Ser190Glu Ala186Glu + Ser187Asp + Phe188Ile + Ser190Glu Ala186Glu + Ser187Glu + Phe188Val + Ser190Glu Ala186Asp + Ser187Asp + Phe188Cys + Ser190Asp Ala186Asp + Ser187Glu + Phe188Val + Ser190Glu Ala186Asp + Ser187Glu + Phe188Ser + Ser190Asp Ala186Asp + Ser187Asp + Phe188Ala + Ser190Asp Ala186Asp + Ser187Asp + Phe188Gly + Ser190Glu Ala186Asp + Ser187Asp + Phe188Thr + Ser190Glu Ala186Glu + Ser187Glu + Phe188His + Ser190Glu Ala186Glu + Ser187Asp + Phe188Pro + Ser190Glu Ala186Glu + Ser187Glu + Phe188Ile + Ser190Glu Ala186Glu + Ser187Glu + Phe188Pro + Ser190Glu Ala186Glu + Ser187Glu + Phe188Gly + Ser190Asp Ala186Glu + Ser187Glu + Phe188Pro + Ser190Asp Ala186Glu + Ser187Asp + Phe188Ser + Ser190Glu Ala186Asp + Ser187Asp + Phe188Tyr + Ser190Glu Ala186Asp + Ser187Glu + Phe188His + Ser190Glu Ala186Ser + Ser187Glu + Phe188Ser + Ser190Glu Ala186His + Ser187Glu + Phe188Ser + Ser190Glu Ala186Ser + Ser187Asp + Phe188Cys + Ser190Glu Ala186Ser + Ser187Glu + Phe188Asn + Ser190Glu Ala186Gln + Ser187Glu + Phe188Ala + Ser190Asp Ala186Gln + Ser187Asp + Phe188Met + Ser190Glu Ala186Asn + Ser187Glu + Phe188His + Ser199Asp Ala186Thr + Ser187Glu + Phe188Ala + Ser190Glu Ala186Pro + Ser187Asp + Phe188Cys + Ser190Glu Ala186Pro + Ser187Asp + Phe188Leu + Ser190Asp Ala186Asn + Ser187Asp + Phe188Gln + Ser190Glu

TABLE 22 Loop 6 - Single Mutation Variants Ala199Asn Ala199Asp Ala199Gln Ala199Glu Ala199Gly Ala199His Ala199Pro Ala199Ser Ala199Thr Pro200Asn Pro200Asp Pro200Gln Pro200Glu Pro200Gly Pro200Ser Gly201Asn Gly201Asp Gly201Gln Gly201Glu Gly201Pro Gly201Ser Val202Ala Val202Asn Val202Asp Val202Cys Val202Gln Val202Glu Val202Gly Val202His Val202Met Val202Pro Val202Ser Val202Thr Ser203Asp Ser203Glu Val204Ala Val204Asn Val204Asp Val204Cys Val204Gln Val204Glu Val204Gly Val204His Val204Met Val204Pro Val204Ser Val204Thr Tyr205Ala Tyr205Asn Tyr205Asp Tyr205Cys Tyr205Gln Tyr205Glu Tyr205Gly Tyr205His Tyr205Ile Tyr205Leu Tyr205Met Tyr205Pro Tyr205Ser Tyr205Thr Tyr205Val Ser206Asp Ser206Glu Thr207Asn Thr207Asp Thr207Gln Thr207Glu Thr207Gly Thr207Pro Thr207Ser Tyr208Ala Tyr208Asn Tyr208Asp Tyr208Cys Tyr208Gln Tyr208Glu Tyr208Gly Tyr208His Tyr208Ile Tyr208Leu Tyr208Met Tyr208Pro Tyr208Ser Tyr208Thr Tyr208Val Pro209Asn Pro209Asp Pro209Gln Pro209Glu Pro209Gly Pro209Ser Ser210Asp Ser210Glu Asn211Asp Asn211Gln Asn211Glu Asn211Ser Thr212Asn Thr212Asp Thr212Gln Thr212Glu Thr212Gly Thr212Pro Thr212Ser Tyr213Ala Tyr213Asn Tyr213Asp Tyr213Cys Tyr213Gln Tyr213Glu Tyr213Gly Tyr213His Tyr213Ile Tyr213Leu Tyr213Met Tyr213Pro Tyr213Ser Tyr213Thr Tyr213Val Thr214Asn Thr214Asp Thr214Gln Thr214Glu Thr214Gly Thr214Pro Thr214Ser Ser215Asp Ser215Glu Leu216Ala Leu216Asn Leu216Asp Leu216Cys Leu216Gln Leu216Glu Leu216Gly Leu216His Leu216Ile Leu216Met Leu216Pro Leu216Ser Leu216Thr Leu216Val Asn217Asp Asn217Gln Asn217Glu Asn217Ser Gly218Asn Gly218Asp Gly218Gln Gly218Glu Gly218Pro Gly218Ser Thr219Asn Thr219Asp Thr219Gln Thr219Glu Thr219Gly Thr219Pro Thr219Ser

TABLE 23 Loop 6 - Double Mutation Variants Val204Ser + Ser210Glu Pro200Gly + Thr214Gly Thr212Asn + Thr219Gly Thr207Ser + Asn211Glu Val204Asn + Tyr208Thr Ser215Asp + Asn217Gln Pro200Gly + Ser215Glu Tyr208Gln + Tyr213Glu Pro200Gln + Leu216Glu Thr207Gln + Ser215Asp Tyr205Gly + Thr219Gly Tyr208Gly + Asn211Glu Val202Cys + Gly218Asn Tyr205Ser + Tyr213Leu Ala199Asn + Gly218Pro Thr207Gln + Thr214Glu Asn211Gln + Ser215Asp Ala199Gly + Gly218Glu Pro200Asn + Thr214Glu Val204Thr + Pro209Asp Pro209Gly + Tyr213Cys Thr214Gln + Asn217Glu Pro200Gln + Pro209Glu Asn211Asp + Thr219Gly Ser203Asp + Val204Gln Tyr205Val + Gly218Glu Tyr205Gln + Thr219Glu Ser203Asp + Leu216Ala Ser210Asp + Gly218Pro Val202Ala + Val204Ser Ala199Pro + Val204Ala Tyr205Gln + Tyr213Gln Tyr205Leu + Pro209Asp Val204Ser + Tyr208Met Thr212Asp + Tyr213Met Val202Ser + Asn211Asp Val202Asp + Tyr213Ser Ser203Asp + Tyr205Ser Tyr213His + Thr219Glu Val202Pro + Asn217Glu Val202Ser + Ser210Asp Val202Thr + Ser215Asp Tyr205Ser + Tyr208Pro Val204Gly + Ser210Glu Thr212Glu + Thr219Gln Tyr208Ala + Gly218Glu Pro200Gly + Tyr213Asp Thr207Pro + Asn211Asp Pro209Gly + Tyr213Ala Val204Met + Ser210Asp Thr214Pro + Asn217Ser Val204Gln + Gly218Pro Tyr205Pro + Thr214Glu Ala199Gln + Ser215Asp Ala199Pro + Ser203Asp Gly201Pro + Thr212Ser Tyr205Pro + Tyr213His Val204Gln + Thr207Ser Thr207Gly + Thr219Asn Pro200Gln + Thr214Pro Val202Cys + Ser215Asp Gly201Pro + Thr214Ser Val204Ser + Pro209Glu Ser203Asp + Asn211Gln Val202Met + Thr212Pro Tyr205Gln + Thr207Asn Ala199Gln + Tyr213Met Gly201Ser + Ser210Asp Gly201Ser + Thr212Asn Tyr208His + Pro209Gly Pro209Gln + Ser215Glu Tyr208Pro + Leu216Thr Tyr213Asp + Thr219Gln Pro209Gln + Thr212Gly Tyr208Met + Pro209Gly Pro209Gly + Asn211Asp Asn211Asp + Tyr213Ser Val202Gln + Ser210Glu Ser203Glu + Thr207Gln Pro200Gln + Val204Thr Asn211Asp + Thr214Ser Ser210Asp + Thr212Gln Val204Gln + Thr219Glu Val202Gln + Tyr205His Thr214Glu + Gly218Pro Pro209Asn + Asn211Ser Ala199Gln + Asn217Glu Pro200Gly + Asn211Ser Gly201Pro + Tyr208Val Val202Asn + Tyr208Cys Val202Gln + Ser215Asp Ala199Pro + Thr219Ser Gly218Pro + Thr219Glu Ala199Gln + Pro200Ser Thr207Pro + Asn217Asp Val204Ser + Asn217Gln Ser203Asp + Val204Met Ser203Asp + Thr219Pro Thr212Pro + Thr219Pro Tyr205Ser + Ser210Glu Ala199His + Gly201Gln Pro209Gly + Ser210Glu Tyr208Gln + Leu216Asn Gly201Ser + Tyr205Leu Val202Met + Thr214Glu Tyr205Ser + Pro209Asp Pro209Ser + Thr212Glu Tyr205Gly + Asn217Asp Pro200Ser + Leu216Ser Ala199His + Tyr208His Asn211Glu + Thr219Asn Ala199Asn + Ser210Glu Tyr205Val + Tyr208His Thr207Ser + Asn211Asp Gly201Ser + Thr207Ser Thr207Asn + Tyr208Val Tyr205Glu + Asn217Gln Tyr208Pro + Thr212Glu Val204Thr + Asn211Glu Tyr205Cys + Asn217Asp Leu216Glu + Thr219Ser Tyr208Ala + Thr212Pro Ala199Ser + Ser203Glu Val204His + Thr212Ser Val204Gln + Gly218Ser Ala199Asn + Ser203Glu Pro200Gln + Val202Met Ala199Asn + Ser203Asp Tyr205Pro + Leu216Asp Tyr205Gln + Ser210Asp Ser210Asp + Asn211Gln Ala199Pro + Asn217Asp Ala199Gly + Asn211Asp Tyr205Ile + Leu216Met Tyr205Gly + Thr214Asn Pro209Glu + Thr219Ser Pro200Gln + Ser210Glu Ala199Gly + Pro200Asn Tyr205Asp + Tyr213Val Ser203Asp + Asn217Gln Val204Met + Thr219Ser Val202Thr + Gly218Ser Pro200Gly + Tyr205Cys Thr207Gly + Thr219Asp Ser215Glu + Gly218Pro Gly201Pro + Tyr205Met Tyr205Ser + Asn217Asp Val202Ser + Pro209Gln Pro200Gly + Gly218Asn Tyr208Ser + Pro209Asp Pro209Gln + Ser215Asp Ala199Thr + Asn217Glu Thr214Asn + Leu216Asp Ser210Asp + Tyr213Val Thr207Asn + Gly218Asp Thr212Pro + Thr219Glu Tyr213Asn + Leu216Glu Gly201Pro + Thr212Asp Gly201Gln + Asn211Ser Val202Met + Leu216Ala Ser215Asp + Leu216Gly Gly201Gln + Leu216Cys Leu216Gly + Thr219Ser Val204Gln + Thr214Gln Thr207Ser + Ser215Glu Thr207Gly + Asn217Glu Pro200Asn + Tyr213Ala Thr212Asp + Thr219Pro Tyr205Glu + Leu216Ile Val202Asp + Tyr208Gly Tyr208Cys + Pro209Asp Thr207Asn + Tyr208Ile Pro200Gly + Ser210Asp Asn211Glu + Thr212Pro Ala199His + Leu216Ser Val204Thr + Asn211Asp Gly218Ser + Thr219Gly Ser203Glu + Val204Ala Val202Thr + Thr219Gly Thr212Asp + Asn217Gln

TABLE 24 Loop 6 - Triple Mutation Variants Thr207Ser + Thr212Asn + Thr219Gly Ala199Asn + Thr207Ser + Asn211Glu Pro200Gln + Leu216Glu + Gly218Asn Tyr205Gly + Thr207Gln + Ser215Asp Tyr208Gly + Asn211Glu + Thr219Gly Val204Thr + Pro209Asp + Tyr213Cys Val204Asn + Thr212Gly + Asn217Glu Pro200Ser + Tyr208Ser + Thr214Gly Ala199Ser + Gly201Ser + Thr214Ser Ala199Gly + Val202Met + Ser210Glu Gly201Asn + Tyr205His + Thr214Gln Ala199Thr + Asn211Asp + Gly218Ser Thr207Gln + Tyr213Gln + Leu216Met Ala199Gly + Val202Cys + Ser210Asp Val204Gln + Tyr213Ala + Thr214Pro Tyr213Leu + Asn217Ser + Thr219Glu Pro200Gly + Thr214Pro + Asn217Asp Pro200Gly + Leu216Val + Thr219Gln Pro200Gly + Gly201Ser + Ser210Asp Gly201Gln + Val202His + Thr212Glu Tyr208His + Asn211Ser + Asn217Asp Thr207Ser + Ser210Asp + Gly218Ser Thr212Asn + Leu216Asn + Thr219Glu Val202Pro + Val204Met + Pro209Asp Val202Asn + Thr212Pro + Thr214Glu Tyr208Ile + Thr212Asp + Tyr213His Ser210Asp + Thr212Ser + Leu216Ala Pro200Gln + Thr214Pro + Leu216His Tyr208Met + Pro209Asp + Leu216Ile Val202Met + Leu216Gly + Thr219Gly Pro200Ser + Tyr205Val + Leu216Cys Val204Thr + Tyr205Glu + Asn217Gln Thr212Glu + Leu216Ile + Asn217Ser Val204Thr + Tyr208Pro + Asn211Glu Thr207Gly + Leu216Glu + Thr219Ser Ser203Glu + Tyr208Ala + Thr212Pro Ala199Ser + Val204His + Thr212Ser Ala199Asn + Val204Gln + Gly218Ser Pro200Gln + Thr214Pro + Gly218Asp Ala199Asn + Val202Met + Ser203Asp Pro200Gln + Ser210Asp + Asn211Gln Tyr205Ile + Thr214Asn + Leu216Met Pro200Gln + Pro209Glu + Thr219Ser Ala199Gly + Pro200Asn + Ser210Glu Val204Met + Asn217Gln + Thr219Ser Val202Thr + Thr214Asp + Gly218Ser Gly201Pro + Tyr205Met + Gly218Pro Val202Ser + Tyr205Ser + Asn217Asp Pro200Gly + Pro209Gln + Gly218Asn Ala199Thr + Thr214Asn + Asn217Glu Thr207Asn + Thr212Pro + Gly218Asp Gly201Pro + Asn211Ser + Thr212Asp Gly201Gln + Ser215Asp + Leu216Gly Val204Gln + Leu216Cys + Thr219Ser Thr207Ser + Thr214Gln + Ser215Glu Pro200Asn + Thr207Gly + Asn217Glu Thr212Asp + Tyr213Ala + Thr219Pro Thr207Asn + Tyr208Ile + Pro209Glu Ala199His + Asn211Glu + Thr212Pro Val204Thr + Asn211Asp + Leu216Ser Ser203Glu + Val204Ala + Thr219Gly Thr207Gly + Thr212Gly + Leu216His Gly201Asn + Thr207Gly + Tyr208Gly Gly201Asn + Val202Glu + Thr207Ser Thr207Gln + Leu216Glu + Gly218Ser Tyr205His + Ser210Asp + Thr219Pro Pro200Gly + Tyr208Val + Ser210Asp Ala199Ser + Ser203Asp + Tyr208Gln Gly201Ser + Asn211Glu + Tyr213Pro Ala199Gln + Tyr205His + Thr219Glu Pro200Ser + Thr207Pro + Thr214Ser Pro200Gln + Tyr205Met + Thr212Pro Ala199Gly + Va 204His + Asn211Glu Thr207Asn + Thr212Ser + Thr219Asn Tyr205Pro + Thr207Gln + Ser215Glu Val204His + Gly218Glu + Thr219Gly Tyr205Pro + Tyr208Ile + Ser215Glu Ala199Asn + Pro200Ser + Ser203Glu Tyr208Gln + Tyr213Met + Thr219Glu Val202Glu + Val204Thr + Asn217Ser Val204Gln + Pro209Gln + Thr214Gly Thr214Gln + Ser215Asp + Leu216Gly Val204Thr + Tyr213Ile + Ser215Glu Val204Gly + Asn211Gln + Ser215Glu Pro200Ser + Val204Gly + Tyr213Ile Pro200Gly + Thr212Asp + Leu216His Asn211Glu + Tyr213Thr + Leu216Pro Leu216Asn + Asn217Asp + Gly218Ser Gly201Gln + Pro209Asn + Ser215Glu Gly201Asn + Ser203Glu + Pro209Asn Gly201Asn + Val202Ser + Leu216Gly Asn211Asp + Tyr213His + Thr219Gln Thr207Gln + Asn217Asp + Gly218Ser Tyr208Gly + Ser210Glu + Thr214Gly Thr207Gln + Tyr208Gln + Tyr213Asp Ala199Asn + Tyr205Ile + Gly218Pro Pro200Asn + Val202Gln + Val204Ser Val204Asn + Ser215Glu + Leu216Pro Pro200Ser + Gly201Ser + Tyr208Ala Tyr208Thr + Tyr213Val + Leu216Asp Thr207Gly + Thr212Ser + Tyr213Ala Gly201Asn + Gly218Asp + Thr219Asn Ala199Gly + Tyr205Cys + Tyr208Thr Gly201Gln + Tyr213Cys + Thr214Gly Ala199Gln + Pro200Ser + Thr214Asp Ala199Ser + Gly201Asn + Thr219Pro Val204Gly + Leu216Val + Thr219Gln Gly201Asn + Ser203Glu + Thr212Gly Gly201Ser + Ser203Glu + Asn217Gln Pro200Gly + Thr207Gln + Asn217Asp Ala199Thr + Tyr208Gln + Thr219Pro Thr207Ser + Thr212Ser + Gly218Asp Pro200Ser + Tyr208Ile + Asn217Ser Val204Pro + Tyr205Asp + Thr214Pro Gly201Ser + Tyr205Glu + Thr207Asn Pro200Ser + Thr207Gln + Asn217Gln Gly201Asn + Val202Glu + Leu216Cys Ala199Ser + Pro209Ser + Gly218Asp Gly201Pro + Tyr205Met + Asn211Asp Gly201Ser + Thr214Pro + Ser215Asp Val204Pro + Thr212Gly + Tyr213Val Gly201Pro + Tyr208Ser + Thr212Glu Ala199Gln + Ser203Asp + Leu216Ser Val202Gly + Asn211Glu + Tyr213Leu Ser203Asp + Thr212Gly + Thr214Gln Gly201Pro + Tyr205Gln + Thr207Gly Gly201Asn + Tyr208Asn + Tyr213Pro Pro200Ser + Tyr213Asp + Gly218Pro Ala199Asn + Pro200Gly + Thr214Ser Ser210Glu + Tyr213Gln + Gly218Asn Ala199Pro + Tyr213Thr + Ser215Asp Val202Pro + Tyr208Cys + Asn211Gln Ala199Gln + Ser203Glu + Asn211Ser Thr207Gln + Pro209Ser + Ser210Glu Pro200Ser + Val202Asn + Gly218Ser Tyr205Val + Tyr213Asn + Gly218Glu Val202Glu + Val204His + Tyr205Gln Ala199Gly + Thr207Pro + Asn211Glu Val202Glu + Tyr208Met + Pro209Gly Val204Ser + Tyr208Ile + Leu216Met Tyr205Thr + Tyr208Ser + Asn217Ser Thr207Ser + Pro209Ser + Leu216Asn Gly201Asn + Pro209Asp + Tyr213Met Gly201Asn + Pro209Gln + Thr214Asn Pro200Asn + Val202Gln + Thr207Gln Ala199Asn + Ser203Glu + Val204Gln Ser203Asp + Pro209Gly + Leu216Ala Ala199Thr + Pro200Asn + Ser210Glu Ala199Thr + Tyr205Leu + Asn217Glu Gly201Asn + Val202Met + Thr214Asp Thr212Gln + Thr214Glu + Thr219Ser Tyr205Glu + Tyr208Gly + Tyr213Leu Pro200Ser + Asn217Glu + Gly218Gln Ser210Glu + Thr214Pro + Leu216Asn Tyr208Asn + Pro209Asn + Tyr213Ser Ala199Pro + Gly201Pro + Ser210Asp Gly201Gln + Thr207Gly + Gly218Glu Pro200Gln + Tyr208Gly + Ser215Asp Pro200Ser + Thr214Pro + Leu216Glu Ala199Gln + Asn217Asp + Gly218Pro Pro200Asn + Val202Asp + Pro209Ser Thr212Gly + Tyr213Asn + Ser215Asp Gly201Gln + Tyr205Met + Thr219Glu Pro200Gly + Tyr213Gly + Thr219Glu Pro200Gly + Ser203Glu + Thr214Gln Tyr208Thr + Ser215Glu + Gly218Ser Ser210Asp + Asn211Ser + Leu216Asn Asn211Gln + Thr214Glu + Ser215Asp Pro209Glu + Ser210Asp + Leu216Cys Tyr208Pro + Asn217Asp + Gly218Asp Tyr213Cys + Ser215Asp + Leu216Asp Thr212Asp + Tyr213Asp + Gly218Ser Ser210Asp + Asn211Glu + Tyr213Gln Val204Cys + Ser210Glu + Asn211Glu Tyr205Glu + Tyr208Ala + Ser215Glu Val202Glu + Ser203Glu + Asn217Asp Pro200Gln + Ser203Glu + Ser215Asp Ala199Asn + Ser203Glu + Asn217Glu Ser203Asp + Tyr208Cys + Asn217Glu Tyr205Asp + Asn211Gln + Thr214Glu

TABLE 25 Loop 6 - Quadruple Mutation Variants Gly201Asn + Tyr205Gly + Thr207Gln + Tyr213Ile Val202Cys + Tyr205Ser + Thr212Pro + Gly218Asn Ala199Ser + Tyr213His + Thr214Gln + Leu216Asn Ala199Ser + Pro200Ser + Tyr208Ser + Thr214Ser Ala199Gly + Gly201Ser + Val202Met + Ser210Glu Gly201Asn + Tyr205His + Thr207Gln + Thr214Gln Val202Cys + Thr207Gln + Tyr213Gln + Leu216Met Val204Gln + Tyr205Asp + Tyr213Ala + Thr214Pro Pro200Gly + Tyr213Leu + Thr214Pro + Asn217Ser Pro200Gly + Leu216Val + Asn217Asp + Thr219Gln Ala199Thr + Val204Ala + Asn217Gln + Gly218Gln Gly201Gln + Val204Gln + Thr207Gln + Leu216His Pro200Asn + Val202Pro + Thr207Gly + Ser215Asp Ala199Ser + Gly201Pro + Ser215Asp + Leu216Ser Ala199Thr + Tyr205Gly + Ser215Gln + Asn217Ser Ala199Gly + Thr207Gln + Tyr208Thr + Pro209Gln Ala199His + Pro200Gly + Gly218Gln + Thr219Gln Ala199Gly + Thr207Asn + Asn211Ser + Thr212Ser Pro200Ser + Val202Gln + Tyr208Ile + Thr219Pro Pro200Gln + Tyr208Met + Pro209Asp + Leu216His Ser203Asp + Asn211Gln + Leu216Thr + Gly218Gln Ala199Gly + Gly201Ser + Tyr205Asp + Leu216Gly Ala199Gln + Gly201Asn + Tyr205Gln + Thr214Asp Pro200Ser + Gly201Pro + Val204Asn + Tyr208Pro Gly201Pro + Val202Gly + Ser210Asp + Leu216Met Pro200Gly + Val202Cys + Ser203Asp + Leu216Pro Tyr205Gly + Thr207Pro + Asn211Gln + Asn217Ser Gly201Pro + Thr207Gln + Thr212Ser + Asn217Asp Pro200Gln + Tyr208Leu + Tyr213Met + Leu216Gln Tyr205Met + Asn211Gln + Tyr213Met + Thr219Asp Val202Met + Tyr205Leu + Tyr208Thr + Tyr213Gly Ser203Asp + Val204Ser + Tyr213Gly + Gly218Pro Gly201Gln + Val202Met + Ser215Asp + Leu216Ala Val204Gln + Thr214Gln + Leu216Cys + Thr219Ser Pro200Asn + Thr212Asp + Tyr213Ala + Thr219Pro Ala199His + Val204Thr + Asn211Asp + Leu216Ser Val202Thr + Ser203Glu + Val204Ala + Thr219Asn Thr207Gly + Tyr208Gly + Thr212Gly + Leu216His Gly201Asn + Ser203Glu + Thr207Gly + Asn211Gln Tyr205His + Thr207Gln + Leu216Gly + Gly218Ser Ala199Gln + Tyr205His + Tyr213Pro + Thr219Glu Ala199Gly + Tyr205Met + Asn211Glu + Thr212Pro Gly201Pro + Thr207Asn + Thr212Gln + Thr219Asn Pro200Gln + Leu216Thr + Asn217Glu + Thr219Pro Gly201Ser + Tyr205Cys + Thr214Asn + Leu216Thr Gly201Gln + Val204Pro + Asn211Asp + Gly218Pro Ser203Asp + Tyr205Val + Thr207Ser + Leu216Cys Val202His + Pro209Gln + Ser215Asp + Leu216Gly Pro200Asn + Gly201Gln + Val202Pro + Ser203Glu Pro200Ser + Gly201Gln + Val202His + Leu216Thr Pro200Asn + Gly201Asn + Leu216Ala + Gly218Pro Ala199Pro + Val202Ser + Val204His + Thr214Glu Gly201Gln + Ser203Glu + Tyr213Ile + Gly218Asn Ala199His + Ser203Asp + Val204Gly + Asn211Gln Pro200Gln + Val202Ala + Val204Pro + Thr207Pro Gly201Asn + Thr212Ser + Tyr213Ala + Gly218Asp Gly201Gln + Tyr213Cys + Thr214Gly + Gly218Glu Ala199Gln + Pro200Ser + Gly201Asn + Thr214Asp Ala199Ser + Val204Gly + Leu216Val + Thr219Pro Pro200Gly + Val202His + Thr207Gln + Asn217Asp Ala199Thr + Tyr208Gln + Thr212Ser + Thr219Pro Pro200Ser + Thr207Ser + Tyr208Ile + Gly218Asp Val204Pro + Tyr205Asp + Thr214Pro + Asn217Ser Gly201Pro + Tyr205Met + Asn211Asp + Thr214Pro Gly201Ser + Tyr213Val + Thr214Pro + Ser215Asp Gly201Pro + Tyr208Ser + Thr212Gly + Tyr213Ala Val202Asn + Asn211Glu + Tyr213Leu + Thr219Pro Gly201Pro + Tyr205Gln + Thr207Gly + Thr212Gly Pro200Gln + Tyr208Cys + Ser215Glu + Thr219Gly Gly201Asn + Tyr208Asn + Tyr213Pro + Ser215Glu Pro200Ser + Gly201Pro + Tyr213Asp + Gly218Pro Ala199Pro + Pro200Ser + Thr212Asp + Thr219Ser Pro200Asn + Ser203Asp + Thr207Gln + Thr212Gly Val204Cys + Pro209Ser + Ser215Asp + Gly218Gln Val202Gln + Val204Ser + Thr212Asp + Asn217Gln Ala199Asn + Pro209Gly + Asn211Glu + Thr214Pro Ala199Asn + Val204Thr + Ser210Asp + Tyr213Leu Pro200Asn + Val204Met + Thr207Gln + Tyr213Asp Val204Gln + Pro209Gly + Thr212Gly + Tyr213Cys Pro209Asn + Tyr213Ile + Thr214Gly + Leu216Met Pro200Gly + Ser203Asp + Leu216Cys + Asn217Ser Val204Ser + Tyr208Ile + Asn211Asp + Leu216Met Gly201Asn + Tyr205Ile + Pro209Gln + Leu216Val Ser203Glu + Val204Gln + Tyr213Leu + Gly218Asn Pro200Asn + Ser210Asp + Thr214Ser + Leu216Cys Ala199Thr + Pro200Asn + Ser210Glu + Leu216Cys Ala199Thr + Val202Met + Tyr205Leu + Thr214Asp Gly201Asn + Thr212Gln + Thr214Glu + Thr219Ser Gly201Pro + Tyr208Asn + Pro209Asn + Tyr213Ser Ala199Pro + Gly201Gln + Thr207Gly + Ser210Asp Ala199Gly + Pro200Gln + Tyr208Asn + Leu216Thr Pro200Asn + Val202Asp + Pro209Ser + Gly218Pro Pro200Gly + Thr207Asn + Leu216Asn + Thr219Asp Ala199Ser + Pro200Gly + Val202Asp + Thr207Ser Val204Ala + Thr207Asn + Ser210Glu + Tyr213Met Pro200Gln + Gly201Ser + Tyr205Thr + Ser215Glu Ala199His + Gly201Asn + Tyr208Ala + Ser210Asp Pro200Gln + Val204Met + Tyr205Ala + Pro209Glu Val202Ala + Tyr205Leu + Thr214Glu + Thr219Gln Thr207Gln + Pro209Asn + Ser210Glu + Thr214Pro Gly201Asn + Ser203Asp + Tyr208Val + Asn211Gln Ala199Ser + Pro200Gly + Leu216Val + Gly218Pro Gly201Gln + Asn211Gln + Thr212Glu + Leu216Ala Val202Cys + Thr207Asn + Tyr213Pro + Leu216Asp Ala199Gly + Val202His + Val204Ala + Ser215Glu Ala199Gln + Thr212Pro + Tyr213Glu + Leu216Ala Pro200Gln + Val202Gln + Thr214Gln + Gly218Glu Gly201Gln + Thr207Pro + Tyr208Met + Thr219Pro Ser203Asp + Val204Gln + Tyr208Ser + Leu216Pro Ala199Asn + Ser203Glu + Val204Asn + Tyr208Pro Pro200Gly + Tyr213Met + Leu216Val + Gly218Asn Tyr205Ile + Tyr213Ile + Thr214Asn + Thr219Pro Pro200Gln + Thr207Gly + Ser215Glu + Leu216Met Ala199Ser + Tyr208Ala + Asn211Gln + Thr219Asp Gly201Pro + Val204Ser + Tyr205His + Thr219Asp Val202Met + Thr207Pro + Thr212Gly + Asn217Glu Pro200Gln + Gly201Pro + Tyr208Thr + Ser215Giu Val204Thr + Thr212Gly + Thr214Glu + Gly218Ser Pro200Gln + Tyr208Ala + Tyr213Glu + Asn217Ser Tyr213Met + Thr214Ser + Ser215Asp + Leu216Pro Thr207Ser + Pro209Asp + Thr212Gly + Thr219Gln Thr207Gln + Asn211Gln + Thr214Glu + Ser215Asp Tyr205Ala + Tyr213Met + Thr214Glu + Ser215Asp Tyr205Met + Thr214Asp + Ser215Glu + Asn217Gln Thr207Asn + Thr214Gly + Leu216Glu + Asn217Asp Val202Thr + Val204His + Asn211Glu + Thr212Asp Pro200Gln + Asn211Asp + Thr212Glu + Thr214Ser Asn211Glu + Thr212Glu + Tyr213Ile + Thr214Asn Gly201Asn + Val202Glu + Ser203Glu + Thr212Gly Thr212Asp + Tyr213Asp + Gly218Ser + Thr219Gly Val204Ala + Thr212Glu + Tyr213Glu + Thr214Asn Pro200Gly + Ser210Asp + Asn211Glu + Thr212Pro Pro209Gln + Ser210Asp + Asn211Glu + Thr219Pro Pro200Ser + Val204Met + Ser210Asp + Asn211Glu Ala199Pro + Val202Ala + Ser210Glu + Asn211Glu Pro209Asp + Ser210Asp + Asn211Glu + Thr212Pro Val202Asp + Ser203Asp + Tyr213Gin + Asn217Glu Ser203Asp + Ser215Asp + Leu216Glu + Thr219Asn Pro200Ser + Ser203Glu + Leu216Glu + Asn217Glu Ser203Asp + Pro209Gly + Ser215Glu + Leu216Ala Ser203Glu + Thr214Gln + Ser215Glu + Gly218Ser Gly201Asn + Ser203Asp + Tyr213Ala + Ser215Glu Ser203Glu + Thr207Ser + Tyr213Gly + Ser215Asp Ala199Gln + Ser203Glu + Thr214Pro + Asn217Asp Ser203Glu + Val204Asn + Thr212Gly + Asn217Glu Ser203Asp + Thr207Gln + Tyr208Cys + Asn217Glu Gly201Ser + Ser203Glu + Thr214Pro + Asn217Asp Asn211Glu + Thr212Asp + Tyr213Glu + Thr219Gly Ala199Pro + Ser203Asp + Val204Pro + Leu216Glu Ala199Asn + Pro209Asp + Asn211Glu + Tyr213Ile Pro200Gln + Tyr205Leu + Pro209Glu + Thr212Asp Ser203Asp + Tyr205Glu + Tyr208Gly + Tyr213Leu Ala199Ser + Val202Glu + Leu216Cys + Gly218Asp Pro200Ser + Thr212Pro + Thr214Glu + Leu216Glu Ala199Asn + Val202Pro + Ser215Glu + Asn217Asp Pro200Gly + Thr207Ser + Ser215Glu + Asn217Glu Gly201Asn + Val202Glu + Thr207Ser + Leu216Glu Ala199Asn + Ser203Glu + Tyr205Glu + Asn217Glu Tyr205Glu + Thr214Pro + Ser215Asp + Asn217Glu Gly201Gln + Thr212Ser + Tyr213Glu + Ser215Glu Ala199Ser + Tyr213Asp + Thr214Pro + Ser215Glu Asn211Ser + Ser215Asp + Asn217Glu + Gly218Asp Ser203Asp + Thr212Gln + Ser215Asp + Gly218Asp Val202Glu + Thr212Gly + Ser215Asp + Leu216Gln Val202Asp + Val204Cys + Tyr208Pro + Thr219Glu Ser203Asp + Pro209Gln + Tyr213Thr + Thr214Asp Val202Asp + Tyr205Glu + Tyr208Gly + Leu216Ile Tyr208Gly + Thr214Asp + Asn217Glu + Gly218Asp Pro200Gln + Tyr208Gln + Tyr213Glu + Leu216Glu Gly201Gln + Tyr205Leu + Thr214Glu + Asn217Asp Ser203Asp + Val204Gly + Thr212Glu + Ser215Asp Gly201Gln + Val202Pro + Asn211Glu + Thr214Glu Thr207Gln + Pro209Ser + Asn211Asp + Thr214Glu Tyr205Ala + Ser210Glu + Thr212Asp + Ser215Asp Ala199Gln + Thr214Glu + Leu216Glu + Thr219Glu Thr212Asp + Thr214Glu + Leu216Asn + Asn217Glu Gly201Asn + Asn211Glu + Thr214Asp + Leu216Glu Pro209Asp + Ser215Asp + Leu216Asp + Gly218Gln Gly201Pro + Val202Cys + Ser203Asp + Thr219Asp Ser203Glu + Thr212Gln + Gly218Gln + Thr219Glu

TABLE 26 Loop 6 - Quintuple Substitution Variants Gly201Asn + Thr207Gln + Ser210Glu + Tyr213Ile + Leu216Ile Val202Cys + Tyr205Ser + Thr212Pro + Tyr213Leu + Gly218Asn Ala199Ser + Pro200Ser + Tyr213His + Thr214Gln + Leu216Asn Ala199Ser + Gly201Ser + Tyr208Ser + Ser210Glu + Thr214Ser Ala199G1y + Gly201Asn + Val202Met + Tyr205His + Thr214Gln Pro200Gly + Tyr213Leu + Thr214Pro + Asn217Ser + Thr219Glu Ala199Thr + Tyr205Gln + Thr207Gln + Thr212Asp + Gly218Gln Val204Gly + Tyr205Ala + Tyr208Gly + Asn211Gln + Gly218Asp Ala199Ser + Gly201Pro + Tyr205Gly + Ser215Glu + Leu216Ser Ala199His + Thr207Gln + Tyr208Thr + Gly218Gln + Thr219Gln Ala199Gly + Pro209Gly + Asn211Ser + Thr212Ser + Tyr213Ser Pro200Ser + Val202Gln + Thr207Asn + Tyr208Ile + Thr219Pro Pro200Ser + Gly201Ser + Tyr208Gln + Ser210Glu + Asn217Gln Ala199Gly + Gly201Ser + Val202Gly + Tyr205Asp + Leu216Gly Ala199Gln + Pro200Ser + Gly201Ser + Tyr205Gln + Thr214Asp Ala199Pro + Gly201Pro + Val204Asn + Tyr208Pro + Thr214Gly Pro200Gly + Val202Gly + Ser203Asp + Asn211Ser + Leu216Pr9 Tyr205Gly + Thr207Pro + Pro209Asp + Asn211Gln + Asn217Ser Pro200Gln + Val204Ala + Tyr208Leu + Thr212Glu + Tyr213Met Tyr205Met + Asn211Gln + Tyr213Met + Thr214Asn + Thr219Asp Ser203Asp + Val204Ser + Tyr213Gly + Thr214Asn + Gly218Pro Gly201Gln + Val204Gln + Ser215Asp + Leu216Ala + Thr219Ser Gly201Asn + Thr207Gly + Tyr208Gly + Thr212Gly + Leu216His Gly201Asn + Tyr205His + Thr207Ser + Leu216Glu + Gly218Ser Ala199Gln + Pro200Ser + Tyr205His + Thr214Ser + Thr219Glu Pro200Gln + Gly201Gln + Leu216His + Asn217Asp + Gly218Gln Ala199Gly + Val204His + Tyr205Met + Asn211Glu + Thr212Pro Val204His + Tyr205Pro + Tyr208Ile + Gly218Glu + Thr219Gly Pro206Ser + Val202Gln + Val204Gly + Tyr213Ile + Ser215Glu Ala199Ser + Pro200Asn + Asn211Glu + Tyr213Thr + Leu216Pro Ser203Asp + Val204Gly + Asn211Gln + Tyr213Ile + Gly218Asn Ala199Asn + Pro200Gln + Val202Ala + Val204Pro + Thr207Pro Ala199Thr + Gly201Gln + Val202Gly + Ser215Asp + Leu216Ala Thr212Gln + Tyr213Val + Leu216His + Gly218Gln + Thr219Glu Ala199Gln + Pro200Ser + Gly201Asn + Tyr205Ala + Thr214Gly Gly201Asn + Val204Gly + Thr212Gly + Leu216Val + Thr219Pro Pro200Gly + Val202His + Thr207Gln + Tyr208Gln + Asn217Asp Ala199Thr + Thr207Ser + Thr212Ser + Gly218Asp + Thr219Pro Pro200Ser + Val204Pro + Tyr208Ile + Thr214Pro + Asn217Ser Pro200Gln + Val202Ser + Tyr205Ser + Asn211Ser + Ser215Asp Val202Thr + Thr212Gln + Ser215Asp + Asn217Ser + Thr219Asn Val204Pro + Thr212Gly + Tyr213Val + Thr214Pro + Ser215Asp Gly201Ser + Val204His + Tyr208Leu + Thr212Ser + Gly218Glu Ser203Asp + Tyr205Gln + Thr207Gly + Thr212Gly + Thr214Gln Pro200Gln + Tyr208Cys + Thr212Gly + Ser215Glu + Thr219Gly Pro200Ser + Gly201Pro + Val202Ala + Tyr213Asp + Gly218Pro Val204Gly + Ser210Asp + Thr212Asn + Thr214Pro + Thr219Ser Pro200Gly + Ser203Asp + Pro209Asn + Leu216Cys + Asn217Ser Tyr205Thr + Thr207Ser + Tyr208Ser + Pro209Ser + Asn217Ser Gly201Asn + Tyr205Ile + Pro209Gln + Thr214Asn + Leu216Val Ala199Asn + Ser203Glu + Val204Gln + Tyr213Leu + Gly218Asn Ala199Thr + Val202Met + Tyr205Leu + Asn211Ser + Asn217Glu Ala199Pro + Gly201Pro + Pro209Asn + Ser210Asp + Tyr213ser Ala199Ser + Gly201Pro + Pro209Gly + Tyr213Pro + Gly218Pro Pro200Ser + Pro209Gly + Thr212Pro + Thr214Pro + Leu216Glu Gly201Ser + Thr207Gly + Ser210Asp + Asn211Ser + Leu216Ile Ala199Ser + Gly201Gln + Val204Asn + Tyr205Leu + Thr219Asp Thr207Pro + Tyr208Thr + Ser210Glu + Thr212Gly + Asn217Ser Gly201Gln + Tyr205Asn + Thr207Asn + Pro209Asp + Thr212Gly Pro200Asn + Tyr205Glu + Tyr208Met + Tyr213Val + Leu216Ile Tyr205Ala + Tyr213His + Thr214Ser + Asn217Gln + Thr219Gln Ala199Gln + Pro200Gly + Val202Met + Leu216Met + Gly218Pro Pro200Ser + Val204Ala + Ser210Glu + Asn211Gln + Gly218Ser Ala199Ser + Pro200Ser + Val202Asn + Leu216Val + Gly218Pro Pro200Gln + Val202Gln + Tyr213Leu + Thr214Pro + Gly218Glu Pro200Ser + Gly201Gln + Thr207Pro + Gly218Pro + Thr219Pro Ala199Asn + Ser203Asp + Val204Asn + Tyr208Pro + Leu216Pro Tyr205Ile + Tyr213Ile + Thr214Asn + Ser215Glu + Thr219Pro Gly201Ser + Val204His + Asn211Gln + Thr212Gln + Leu216Met Ala199Ser + Gly201Pro + Asn211Glu + Gly218Gln + Thr219Ser Ala199Ser + Thr207Ser + Thr214Gln + Ser215Glu + Leu216Asn Ala199His + Val204Cys + Asn211Ser + Thr214Gly + Thr219Glu Ala199Asn + Val202His + Tyr208Asn + Leu216Thr + Thr219Gly Ser203Glu + Thr207Pro + Tyr208Leu + Thr212Ser + Asn217Gln Pro200Ser + Val202Asp + Val204Asn + Tyr208Met + Thr212Gln Gly201Ser + Thr207Asn + Tyr208Leu + Ser210Asp + Asn217Ser Val202Asp + Val204Gln + Tyr208Cys + Pro209Gln + Tyr213Val Ala199Gly + Val202Thr + Thr207Gln + Thr212Glu + Thr214Gln Gly201Pro + Val202Cys + Ser210Asp + Thr212Pro + Tyr213Cys Ala199Asn + Gly201Ser + Val204Met + Pro209Asn + Thr214Pro Gly201Asn + Ser203Asp + Val204Thr + Thr207Ser + Leu216Ala Val202Thr + Tyr205Gly + Thr207Gln + Asn211Asp + Leu216Ala Ala199Gln + Val204Gly + Thr207Pro + Asn211Asp + Thr214Gly Gly201Pro + Val202Asn + Tyr205Ser + Asn211Glu + Gly218Asn Val204Gly + Pro209Asn + Asn211Asp + Asn217Gln + Thr219Gly Val202Gly + Val204Gly + Ser210Glu + Gly218Ser + Thr219Ser Ala199Ser + Asn211Ser + Tyr213Met + Thr214Asp + Thr219Pro Ala199Asn + Gly201Asn + Ser203Glu + Tyr213His + Leu216Ile Ala199Gln + Pro200Asn + Thr207Ser + Pro209Ser + Asn217Ser Tyr205Met + Thr214Asp + Ser215Glu + Leu216cys + Asn217Gln Ala199Pro + Val202His + Tyr208Met + Thr214Glu + Ser215Asp Pro200Asn + Pro209Glu + Ser210Asp + Thr214Ser + Leu216cys Val204Ser + Tyr208Pro + Leu216Met + Asn217Asp + Gly218Asp Tyr205Pro + Thr207Pro + Leu216Asn + Asn217Asp + Gly218Asp Pro200Gly + Tyr205Asn + Tyr208Gly + Asn217Glu + Gly218Glu Val202Thr + Val204His + Pro209Gly + Asn211Glu + Thr212Asp Gly201Gln + Val202Asn + Thr207Asn + Ser215Glu + Leu216Asp Ala199His + Pro200Gly + Ser210Asp + Asn211Glu + Thr212Pro Pro209Gln + Ser210Asp + Asn211Glu + Asn217Gln + Thr219Pro Ala199Pro + Val202Ala + Ser210Glu + Asn211Asp + Gly218Asn Ala199Asn + Thr207Asn + Ser210Asp + Asn211Asp + Thr212Gly Val204Ala + Tyr205Glu + Thr212Ser + Ser215Glu + Asn217Gln Gly201Pro + Val202Asp + Thr207Gln + Thr212Ser + Asn217Asp Thr207Ser + Pro209Asp + Ser210Glu + Asn211Asp + Tyr213Asn Gly201Ser + Val202Glu + Ser203Glu + Thr214Pro + Asn217Asp Ser203Glu + Thr207Asn + Thr214Gly + Leu216Glu + Asn217Asp Gly201Ser + Ser203Asp + Tyr208Ile + Leu216Asp + Asn217Asp Gly201Pro + Tyr208Gly + Leu216Asp + Asn217Glu + Gly218Asp Ser203Glu + Val204Pro + Thr207Gly + Tyr213Leu + Ser215Asp Gly201Asn + Ser203Asp + Tyr208Val + Asn211Gln + Ser215Asp Ala199Asn + Pro200Ser + Ser203Glu + Tyr213Met + Ser215Glu Ala199Ser + Ser203Glu + Asn211Gln + Ser215Glu + Leu216cys Ser203Glu + Val204Gln + Thr214Gln + Ser215Asp + Leu216Gly Ala199Pro + Gly201Pro + Ser203Asp + Tyr205Val + Ser215Glu Ala199Thr + Ser203Asp + Val204Gln + Tyr213Gly + Ser215Asp Val202Cys + Ser203Asp + Tyr205Met + Tyr213Gln + Asn217Glu Ser203Glu + Val204Asn + Thr212Gly + Asn217Glu + Thr219Gln Gly201Asn + Ser203Asp + Tyr208Val + Pro209Ser + Asn217Glu Ser203Asp + Thr212Pro + Tyr213Gly + Asn217Glu + Gly218Ser Val204Thr + Asn211Asp + Thr212Asp + Tyr213Asp + Leu216Ser Val202Ala + Ser203Asp + Val204Gln + Tyr205Met + Leu216Glu Ser203Glu + Val204Ser + Thr207Gly + Tyr208Val + Leu216Asp Pro200Gln + Pro209Gln + Ser210Glu + Thr212Glu + Thr214Gly Pro209Glu + Asn211Asp + Asn217Gln + Gly218Gln + Thr219Pro Pro200Ser + Tyr205Pro + Pro209Asp + Asn211Glu + Tyr213Ile Ala199Asn + Ser203Asp + Tyr205Glu + Thr207Asn + Leu216Glu Ser203Glu + Tyr205Glu + Tyr213Met + Leu216Ile + Asn217Gln Val202Glu + Ser203Glu + Asn211Gln + Leu216Met + Gly218Glu Ala199Ser + Val2025er + Tyr205Asp + Tyr208Gly + Tyr213Asp Pro200Ser + Thr207Pro + Thr212Asp + Thr214Asp + Thr219Ser Pro200Gly + Thr207Ser + Thr214Gln + Ser215Glu + Asn217Glu Pro200Gln + Thr207Gly + Ser215Asp + Leu216Gln + Asn217Asp Ala199Asn + Tyr205Glu + Tyr208Ala + Ser215Glu + Asn217Glu Gly201Pro + Val204Ser + Tyr205His + Asn217Glu + Thr219Asp Ala199Ser + Tyr208Gln + Tyr213Asp + Thr214Pro + Ser215Glu Thr207Asn + Tyr208Gln + Thr212Ser + Tyr213Glu + Ser215Glu Gly201Gln + Tyr205Gly + Pro209Glu + Thr212Pro + Tyr213Glu Ser203Asp + Pro209Ser + Thr212Gln + Ser215Asp + Gly218Asp Ser203Asp + Pro209Gly + Ser215Glu + Leu216Ala + Gly218Asp Pro200Ser + Ser203Asp + Thr207Gln + Leu216Gln + Gly218Glu Pro200Asn + Gly201Pro + Ser203Glu + Thr207Gly + Gly218Glu Val202Asp + Val204Cys + Tyr208Ala + Gly218Gln + Thr219Glu Val202Glu + Val204Thr + Tyr208Gln + Asn217Ser + Thr219Glu Pro200Ser + Gly201Pro + Val202Asp + Asn211Gln + Thr219Glu Gly201Asn + Ser203Glu + Thr212Gln + Thr214Asp + Thr219Ser Gly201Asn + Ser203Asp + Thr214Glu + Leu216Cys + Thr219Gly Gly201Asn + Val202Cys + Ser203Asp + Thr214Asp + Thr219Pro Val202Cys + Ser203Asp + Tyr208Thr + Thr214Asp + Thr219Pro Val204Ser + Tyr208Leu + Ser210Asp + Tyr213Asp + Thr214Asp Ala199His + Ser203Asp + Tyr208Ser + Tyr213Glu + Thr214Asp Val204Cys + Ser210Asp + Asn211Ser + Tyr213Asp + Thr219Gln Ala199Asn + Ser210Glu + Thr212Asn + Tyr213Glu + Thr214Gln Ser203Asp + Tyr205Glu + Tyr208Gly + Tyr213Leu + Gly218Asp Val202Glu + Tyr205Glu + Pro209Asn + Leu216Thr + Gly218Glu Tyr208Val + Ser215Asp + Asn217Ser + Gly218Asp + Thr219Glu Tyr205Leu + Thr214Asp + Ser215Glu + Gly218Glu + Thr219Asn Ala199Pro + Pro209Asp + Thr212Asp + Ser215Asp + Gly218Ser Ser203Glu + Thr212Glu + Tyr213Ser + Thr214Glu + Leu216Cys Ser203Asp + Tyr208Cys + Thr212Asp + Tyr213Ile + Thr214Asp Val202Met + Val204Thr + Asn211Asp + Thr214Asp + Ser215Asp Pro209Asn + Asn211Asp + Thr214Glu + Ser215Glu + Leu216Gly Pro200Asn + Tyr205Glu + Thr212Asp + Tyr213Ala + Thr219Pro Pro200Asn + Ser203Asp + Tyr205His + Thr212Asp + Ser215Asp Ser203Asp + Val204Thr + Thr212Glu + Tyr213Ile + Ser215Glu Tyr205Gly + Thr207Gln + Tyr208Ile + Thr212Glu + Ser215Glu Ala199Asn + Tyr205Asp + Ser210Glu + Thr212Glu + Thr214Gly Val202Met + Tyr205Ala + Ser210Glu + Thr212Asp + Ser215Asp Thr207Gln + Tyr208Leu + Asn211Glu + Tyr213Glu + Leu216Glu Val204Met + Pro209Ser + Asn211Asp + Thr214Glu + Leu216Asp Pro200Ser + Ser203Glu + Tyr213Glu + Leu216Gly + Asn217Glu Ala199Gly + Tyr213Pro + Thr214Asp + Asn217Asp + Thr219Asp Ala199Gln + Tyr208Ala + Thr214Glu + Asn217Glu + Thr219Asp Ala199His + Val202His + Ser203Glu + Thr207Gly + Thr219Asp Gly201Ser + Val202Gly + Ser203Glu + Tyr205Thr + Thr219Asp Gly201Pro + Pro209Glu + Ser210Asp + Ser215Glu + Leu216Met Tyr205Glu + Ser215Glu + Leu216Cys + Gly218Gln + Thr219Glu Ser203Asp + Pro209Ser + Asn211Glu + Thr214Asp + Leu216Asn Ala199Thr + Ser203Glu + Thr207Gln + Asn211Asp + Thr214Glu Gly201Ser + Ser203Asp + Tyr205Gly + Pro209Asp + Ser215Asp Pro209Glu + Thr214Asn + Leu216Glu + Asn217Asp + Thr219Asn

TABLE 27 Loop 6 - Sextuple Substitution Variants Gly201Asn + Tyr205Gly + Thr207Gln + Ser210Glu + Tyr213Ile + Thr219Gly Pro200Asn + Gly201Ser + Tyr208Gly + Asn211Asp + Thr212Ser + Thr219Asn Gly201Pro + Ser203Asp + Val204Gln + Tyr205Pro + Thr212Ser + Tyr213His Pro200Gln + Val204His + Thr207Ser + Thr214Pro + Ser215Asp + Thr219Asn Ala199Pro + Pro200Ser + Gly201Pro + Val204Asn + Tyr208Pro + Thr214Gly Pro200Gln + Val204Ala + Tyr208Leu + Thr212Glu + Tyr213Met + Leu216Gln Ala199Pro + Val202Met + Ser203Glu + Tyr205Leu + Asn211Gln + Tyr213Gly Gly201Gln + Val204Gln + Thr214Gln + Ser215Asp + Leu216Gly + Thr219Ser Val204Thr + Tyr208Met + Pro209Glu + Tyr213Gly + Thr214Pro + Gly218Ser Ser203Asp + Val204Met + Thr207Gln + Pro209Gln + Asn217Ser + Thr219Asn Pro200Ser + Val202Met + Val204Gly + Pro209Gln + Tyr213Ile + Ser215Glu Ala199His + Ser203Asp + Val204Gly + Thr207Gly + Asn211Gln + Thr214Ser Ala199Gln + Pro200Gln + Gly201Gln + Pro209Glu + Leu216Ile + Asn217Ser Pro200Gln + Tyr20811e + Pro209Gln + Thr212Gly + Ser215Glu + Thr219Gly Pro200Asn + Ser203Asp + Val204Cys + Thr207Gln + Pro209Ser + Thr214Asn Ala199Asn + Val202Gln + Val204Ser + Pro209Gly + Thr212Asp + Asn217Gln Pro200Asn + Val204Met + Thr207Gln + Pro209Gly + Thr212Gly + Tyr213Asp Pro200Gly + Val204Gln + Pro209Asn + Tyr213Ile + Thr214Gly + Leu216Met Ala199Asn + Tyr205Thr + Tyr208Ser + Pro209Ser + Thr214Asp + Asn217Ser Tyr205Ser + Thr207Gln + Tyr208Cys + Pro209Asn + Thr212Asn + Leu216Glu Ala199Asn + Pro200Ser + Ser203Asp + Tyr205Cys + Tyr208Asn + Thr214Gln Thr207Pro + Tyr213Ser + Thr214Asn + Asn217Ser + Gly218Ser + Thr219Glu Ala199Pro + Gly201Pro + Thr207Gly + Pro209Asn + Ser210Asp + Tyr213Ser Ala199Gln + Pro200Gly + Val202Met + Thr212Asp + Leu216Met + Gly218Pro Ala199Ser + Pro200Gly + Val202Asn + Asn211Gln + Leu216Val + Gly218Pro Ala199His + Pro200Gln + Val202Gln + Thr214Gln + Leu216Thr + Gly218Glu Pro200Ser + Gly201Gln + Thr207Pro + Tyr208Thr + Gly218Pro + Thr219Pro Pro200Gln + Val204Met + Thr207Gly + Ser215Glu + Leu216Met + Thr219Pro Thr207Ser + Pro209Asp + Thr212G1y + Thr214Ser + Leu216Pro + Thr219Gln Pro200Asn + Gly201Ser + Thr207Asn + Tyr208Leu + Ser210Asp + Asn217Ser Ala199Asn + Val202Ser + Tyr205Cys + Thr207Gln + Tyr213Leu + Asn217Ser Val202Ser + Pro209Gln + Tyr213Asp + Leu216Met + Asn217Ser + Thr219Gly Gly201Gln + Val204Gly + Pro209Asn + Asn211Asp + Asn217Gln + Thr219Gly Val202Gly + Ser203Glu + Val204Gly + Tyr213Met + Gly218Ser + Thr219Ser Ala199Gln + Pro200Asn + Tyr205Thr + Thr207Ser + Thr214Gln + Asn217Ser Ala199Ser + Gly201Asn + Val202Met + Val204Asn + Asn211Gln + Gly218Glu Pro200Asn + Val2d2Pro + Val204Gln + Tyr208Pro + Gly218Gln + Thr219Glu Thr207Gly + Tyr208Ile + Pro209Gly + Tyr213Gly + Thr214Pro + Leu216Asn Val202Met + Ser203Asp + Val204Met + Thr212Asn + Thr214Asn + Leu216Gly Ala199Ser + Val202Met + Val204Ala + Thr212Gly + Tyr213Thr + Thr214Pro Tyr208Val + Pro209Gln + Thr214Ser + Leu216Cys + Asn217Glu + Thr219Asn Pro200Gln + Gly201Gln + Tyr205Ala + Pro209Gln + Leu216Pro + Thr219Glu Ala199Gly + Pro200Gln + Gly201Ser + Tyr208Gln + Ser215Asp + Gly218Asn Ala199Asn + Pro200Asn + Val202Asn + Val204Gln + Thr214Gly + Asn217Glu Pro200Gly + Gly201Gln + Tyr213Gly + Thr214Asn + Leu216Gln + Thr219Ser Gly201Gln + Val202Gly + Thr207Gly + Asn211Gln + Thr214Gly + Ser215Asp Ala199Thr + Pro200Ser + Tyr208Ala + Thr212Ser + Ser215Asp + Asn217Ser Ala199His + Val202Asn + Ser203Glu + Val204His + Tyr213Ile + Asn217Gln Gly201Asn + Val202Cys + Tyr205Cys + Tyr213Ile + Leu216Pro + Asn217Asp Ala199Thr + Gly201Ser + Val202Gly + Tyr205Pro + Leu216Thr + Gly218Glu Ala199Ser + Val204Cys + Tyr208Cys + Pro209Gly + Tyr213Ala + Leu216Asp Ala199Gly + Gly201Asn + Val204Met + Thr207Pro + Thr214Glu + Leu216Met Pro200Gly + Tyr205Met + Tyr208Val + Thr212Gly + Tyr213Ser + Leu216Val Gly201Pro + Tyr208Val + Pro209Glu + Asn211Gln + Tyr213Ala + Thr219Gly Ala199Ser + Pro200Ser + Val202Thr + Tyr205Gln + Gly218Asp + Thr219Pro Ala199Gln + Ser203Glu + Val204Pro + Thr214Ser + Gly218G1n + Thr219Pro Ala199Thr + Pro200Gly + Gly201Gln + Thr212Ser + Leu216Val + Gly218Ser Pro200Ser + Val204Gln + Thr207Pro + Thr214Gly + Ser215Glu + Gly218Gln Ala199Gly + Gly201Gln + Val204His + Tyr205Asp + Pro209Gln + Thr212Ser Pro200Gln + Tyr208Asn + Asn211Ser + Thr212Ser + Tyr213Thr + Leu216Asp Ala199His + Tyr208Leu + Thr212Pro + Ser215Glu + Leu216Ser + Gly218Gln Gly201Pro + Val204Cys + Pro209Gln + Thr212Ser + Tyr213Cys + Thr214Gln Val204Ser + Tyr20SSer + Thr207Asn + Tyr208Cys + Ser215Asp + Asn217Ser Pro200Asn + Tyr208Ala + Tyr213Pro + Thr214Glu + Ser215Asp + Thr219Gly Val202His + Tyr208Met + Pro209Asn + Thr212Asn + Thr214Glu + Ser215Asp Pro200Gln + Gly201Ser + Val202Pro + Val204Ser + Thr214Asp + Ser215Glu Ala199Gln + Gly201Ser + Val204Gly + Thr207Ser + Thr214Asp + Ser215Glu Val202Asn + Tyr205Gly + Pro209Gln + Thr212Ser + Thr214Glu + Ser215Glu Pro200Asn + Pro209Glu + Ser210Asp + Asn211Ser + Thr214Ser + Leu216Cys Pro200Ser + Val204Pro + Tyr208Ile + Thr214Pro + Asn217Asp + Gly218Asp Gly201Gln + Tyr205Asn + Tyr208Gly + Thr212Gly + Asn217Glu + Gly218Glu Val202His + Thr207Asn + Tyr208Gln + Tyr213Pro + Ser215Glu + Leu216Asp Ala199Asn + Gly201Ser + Val202Pro + Tyr205Thr + Ser215Asp + Leu216Asp Pro200Gly + Val202Ala + Val204Ala + Ser210Glu + Asn211Glu + Gly218Ser Val204Asn + Pro209Ser + Ser210Asp + Asn211Asp + Tyr213Asn + Leu216Met Ala199Asn + Val204Thr + Ser210Asp + Asn211Glu + Tyr213Leu + Thr214Pro Val202Cys + Val204Ala + Tyr205Glu + Thr212Ser + Ser215Glu + Asn217Gln Thr207Pro + Ser210Asp + Asn211Glu + Thr212Glu + Asn217Ser + Thr219Ser Pro200Asn + Val202Asp + Tyr205Ala + Pro209Ser + Asn217Asp + Gly218Pro Val202Gln + Pro209Glu + Ser210Glu + Asn211Glu + Thr212Gln + Asn217Gln Val202Asp + Ser203Asp + Thr207Gln + Thr212Ser + Tyr213Gln + Asn217Glu Ala199Asn + Ser203Glu + Val204Asn + Tyr208Pro + Leu216Glu + Asn217Asp Val204Thr + Thr212Gly + Thr214Glu + Ser215Asp + Leu216Glu + Gly218Ser Ala199Gln + Ser203Asp + Val204Cys + Thr207Gly + Ser215Glu + Leu216Gln Ala199Pro + Pro200Gln + Ser203Asp + Asn211Ser + Ser215Asp + Leu216Asn Ser203Glu + Tyr208Thr + Tyr213Cys + Thr214Gln + Ser215Glu + Gly218Ser Ala199Thr + Pro200Gln + Gly201Gln + Ser203Asp + Ser215Asp + Gly218Ser Pro200Gly + Val202Cys + Ser203Asp + Asn211Ser + Ser215Glu + Leu216Pro Ala199Gln + Ser203Glu + Tyr205Gly + Tyr213Met + Ser215Glu + Asn217Ser Pro200Ser + Gly201Asn + Ser203Asp + Tyr208Val + Pro209Ser + Asn217Glu Pro200Ser + Gly201Ser + Val202His + Ser203Asp + Asn217Glu + Gly218Pro Val202Asn + Tyr205Glu + Thr207Gln + Tyr208Leu + Thr214Asp + Gly218Pro Ser203Asp + Val204Thr + Tyr205Glu + Thr212Ser + Tyr213Ile + Ser215Glu Ala199Asn + Pro200Ser + Tyr205Pro + Pro209Asp + Asn211Glu + Tyr213Ile Gly201Pro + Val202Cys + Val204Ser + Pro209Glu + Thr212Glu + Thr214Ser Ala199Asn + Val202Asp + Ser203Asp + Tyr208Cys + Ser215Asp + Leu216Asn Ala199Asn + Ser203Glu + Tyr205Cys + Thr212Gly + Thr214Glu + Ser215Asp Ala199Thr + Pro200Ser + Tyr208Met + Thr214Ser + Ser215Glu + Asn217Asp Ala199Pro + Tyr208Gly + Ser215Asp + Leu216Val + Asn217Asp + Thr219Ser Pro200Asn + Pro209Gly + Tyr213Pro + Ser215Asp + Leu216Gly + Asn217Asp Pro200Ser + Gly201Pro + Val202Ala + Asn211Glu + Tyr213Asp + Gly218Pro Val202Met + Val204Ser + Tyr205His + Thr212Gly + Asn217Glu + Thr219Asp Tyr205Val + Tyr208Pro + Ser215Glu + Leu216Met + Asn217Asp + Gly218Asp Val202Ser + Tyr208Ala + Asn211Ser + Thr214Glu + Leu216Asp + Asn217Asp Gly201Pro + Tyr208Gln + Thr212Asp + Tyr213Asp + Ser215Asp + Asn217Ser Ala199Gln + Gly201Gln + Tyr205Gly + Pro209Glu + Thr212Pro + Tyr213Glu Ser203Asp + Pro209Ser + Asn211Gln + Thr212Gln + Ser215Asp + Gly218Asp Ser203Asp + Pro209Gly + Tyr213Leu + Ser215Glu + Leu216Ala + Gly218Asp Val202Glu + Val204Cys + Thr212Gly + Ser215Asp + Leu216Gln + Gly218Gln Pro200Gly + Gly201Gln + Val202Glu + Val204Ala + Thr207Pro + Ser215Asp Ala199Gln + Pro200Ser + Gly201Ser + Ser203Asp + Gly218Glu + Thr219Asp Ala199Gln + Gly201Ser + Tyr205Gln + Ser210Asp + Thr212Glu + Thr214Asp Gly201Asn + Val202Pro + Ser210Glu + Thr212Glu + Tyr213Thr + Thr214Glu Pro200Gln + Val202Met + Ser203Asp + Val204Gly + Tyr208Ile + Gly218Asp Pro200Ser + Gly201Pro + Val202Asp + Tyr208Cys + Asn211Gln + Thr219Glu Ser203Asp + Tyr208Pro + Pro209Ser + Asn211Ser + Thr214Asp + Thr219Pro Ala199His + Gly201Asn + Ser203Glu + Tyr213His + Thr214Glu + Leu216Ile Ser203Glu + Tyr208Pro + Tyr213His + Thr214Asp + Leu216Cys + Asn217Ser Val202Asp + Tyr205Met + Thr214Asp + Ser215Glu + Leu216Cys + Asn217Gln Val202Cys + Val204Ser + Tyr208Leu + Ser210Asp + Tyr213Asp + Thr214Asp Gly201Asn + Tyr205Gln + Pro209Asn + Ser210Glu + Tyr213Glu + Leu216Ala Val202Cys + Pro209Asp + Tyr213Asp + Thr214Asn + Ser215Glu + Asn217Gln Ala199Gly + Gly201Gln + Ser215Asp + Leu216Asp + Gly218Asn + Thr219Glu Ala199His + Thr207Asn + Asn211Ser + Leu216Glu + Asn217Gln + Thr219Asp Val202Met + Thr207Gln + Pro209Gly + Thr214Glu + Ser215Asp + Gly218Asp Pro200Gln + Val202Ala + Val204Pro + Thr207Gln + Pro209Glu + Thr214Glu Gly201Pro + Tyr208Gly + Thr214Asp + Leu216Cys + Asn217Glu + Gly218Asp Ala199Gln + Thr207Pro + Tyr208Cys + Pro209Gln + Tyr213Asp + Leu216Asp Ser203Asp + Thr214Gly + Ser215Glu + Leu216Ala + Asn217Ser + Thr219Glu Pro200Gln + Ser203Glu + Val204His + Thr212Asp + Thr214Asp + Gly218Gln Ala199His + Gly201Ser + Ser203Glu + Tyr205Ser + Thr214Glu + Gly218Glu Gly201Pro + Ser203Glu + Thr207Gly + Tyr208Cys + Thr214Asp + Gly21eGlu Ser203Glu + Tyr205Leu + Thr207Asn + Thr212Asp + Tyr213Met + Ser215Glu Ala199Pro + Ser203Asp + Pro209Ser + Thr212Glu + Ser215Asp + Gly218Gln Ser203Glu + Tyr205His + Thr212Asp + Tyr213Met + Ser215Asp + Thr219Ser Ser203Asp + Val204Gly + Thr212Glu + Thr214Pro + Ser215Asp + Asn217Ser Gly201Pro + Tyr205Gln + Thr207Gly + Asn211Glu + Thr212Gly + Thr214Glu Pro200Asn + Val202Gln + Thr207Gln + Pro209Ser + Asn211Asp + Thr214Glu Gly201Gln + Thr207Gly + Pro209Gln + Thr212Glu + Ser215Glu + Gly218Gln Val202Met + Tyr205Ala + Ser210Glu + Thr212Asp + Ser215Asp + Leu216Ala Gly201Gln + Pro209Gly + Asn211Asp + Tyr213Asp + Thr214Gly + Leu216Asp Ala199Gly + Val202Glu + Ser203Glu + Tyr213Glu + Thr214Pro + Thr219Ser Gly201Ser + Val202Cys + Ser203Asp + Tyr205Asp + Leu216Gly + Thr219Asp Pro200Ser + Ser203Glu + Tyr213Glu + Leu216Gly + Asn217Glu + Thr219Gly Tyr205Asn + Pro209Asn + Thr212Glu + Tyr213Met + Ser215Glu + Asn217Asp Ala199Gln + Tyr208Ala + Asn211Gln + Thr214Glu + Asn217Glu + Thr219Asp Gly201Pro + Val202Gly + Ser203Glu + Tyr205Thr + Tyr208Ala + Thr219Asp Pro200Asn + Gly201Pro + Ser203Asp + Thr207Gln + Gly218Ser + Thr219Glu Gly201Asn + Ser203Asp + Val204Gly + Tyr208Val + Asn211Gln + Thr219Asp Ala199His + Pro200Asn + Ser203Asp + Tyr205Thr + Pro209Asp + Thr214Glu Gly201Pro + Val202Gly + Pro209Glu + Ser210Asp + Ser215Glu + Leu216Met Ala199Gly + Val202His + Val204Ala + Pro209Asp + Ser210Glu + Ser215Glu Gly201Asn + Val204Gln + Tyr208Leu + Thr214Glu + Ser215Glu + Thr219Glu Ser203Glu + Tyr205Gln + Tyr208Gly + Asn211Glu + Thr214Glu + Thr219Ser Gly201Ser + Ser203Asp + Tyr205Ala + Pro209Asp + Tyr213Met + Ser215Asp Val202Asn + Pro209Asp + Asn211Glu + Thr212Ser + Tyr213Leu + Ser215Glu Ala199Pro + Val204Thr + Tyr205Glu + Ser210Glu + Asn211Asp + Tyr213Leu Val202Ala + Ser203Asp + Val204Gln + Tyr205Met + Thr212Glu + Leu216Glu Gly201Pro + Ser203Asp + Tyr208Thr + Thr214Asp + Asn217Gln + Thr219Asp Pro200Gln + Gly201Asn + Thr207Gln + Tyr213Glu + Asn217Asp + Gly218Asp Pro200Gln + Thr207Gly + Tyr208Ala + Thr212Glu + Tyr213Glu + Asn217Glu Thr207Ser + Tyr208Ser + Pro209Glu + Thr214Glu + Asn217Glu + Thr219Asn Ala199Gly + Tyr205Pro + Pro209Glu + Thr214Glu + Leu216Asn + Asn217Asp Ala199His + Gly201Asn + Tyr205Pro + Pro209Glu + Ser215Glu + Asn217Glu Pro200Gly + Pro209Asp + Tyr213Gln + Ser215Glu + Leu216Ile + Asn217Asp Ala199His + Val204Ser + Thr207Gln + Pro209Asp + Ser210Asp + Thr219Asp Ala199Thr + Thr207Pro + Tyr208Val + Pro209Glu + Ser210Asp + Thr219Asp Tyr208His + Pro209Glu + Ser210Glu + Tyr213Val + Leu216Cys + Thr219Asp Tyr205Met + Thr207Gly + Ser210Asp + Thr212Ser + Asn217Glu + Gly218Asp Ser210Glu + Thr212Pro + Thr214Gln + Asn217Asp + Gly218Glu + Thr219Ser Pro200Gln + Val204Thr + Tyr208Asn + Asn211Asp + Ser215Glu + Leu216Glu Gly201Pro + Tyr208Gln + Asn211Glu + Thr212Asp + Asn217Glu + Thr219Ser Val202Asp + Ser203Asp + Asn211Glu + Thr212Pro + Tyr213Leu + Thr214Asn Val202Glu + Ser203Asp + Tyr205Leu + Pro209Gly + Thr212Asp + Thr219Ser Ala199Gly + Pro200Asn + Tyr208Leu + Ser210Asp + Asn211Asp + Asn217Asp Pro200Ser + Gly201Ser + Val202Asp + Tyr208Gln + Ser210Glu + Asn211Asp Val202Asp + Thr207Gln + Tyr208Thr + Ser210Glu + Thr214Gln + Asn217Glu Thr207Gln + Asn211Asp + Thr214Glu + Leu216His + Asn217Asp + Gly218Gln Ser203Glu + Thr207Ser + Pro209Gly + Asn211Asp + Tyr213Gly + Ser215Asp Ser203Glu + Tyr205Val + Pro209Gly + Asn211Asp + Ser215Asp + Thr219Gly

TABLE 28 Loop 6 - Heptuple Substitution Mutation Variants Ala199Ser + Pro200Ser + Val204Met + Tyr208Ser + Tyr213His + Thr214Gln + Leu216Ala Ala199Ser + Gly201Ser + Val202Met + Thr207Gln + Ser210Glu + Thr214Ser + Asn217Gln Gly201Gln + Val204Gly + Tyr205Ala + Thr207Gln + Tyr208Gly + Asn211Gln + Gly218Asp Ala199Gly + Pro200Gly + Thr207Gln + Tyr208Thr + Pro209Gln + Gly218Gln + Thr219Gln Ala199Gly + Pro200Ser + Val202Gln + Thr207Asn + Asn211Ser + Thr212Ser + Thr219Pro Pro200Gln + Val204Ala + Tyr208Leu + Thr212Glu + Tyr213Met + Thr214Asn + Leu216Gln Ser203Asp + Val204Met + Thr207Gln + Tyr208Met + Pro209Gln + Asn217Ser + Thr219Asn Ala199Gln + Pro200Ser + Gly201Gln + Tyr205Ala + Tyr213Cys + Thr214Gly + Gly218Glu Ala199Thr + Pro200Ser + Thr207Gln + Tyr208Gln + Thr212Ser + Gly218Asp + Thr219Pro Gly201Ser + Val204Pro + Tyr208Ser + Thr212Gly + Tyr213Val + Thr214Pro + Ser215Asp Pro200Gln + Tyr208Cys + Pro209Gln + Thr212Gly + Tyr213Pro + Ser215Glu + Thr219Gly Val204Met + Thr207Gln + Pro209Gly + Thr212Gly + Tyr213Cys + Thr214Gly + Leu216Met Pro200Gly + Ser203Asp + Thr207Asn + Pro209Asn + Tyr213Ile + Leu216Cys + Asn217Ser Pro200Asn + Gly201Asn + Tyr205Ile + Pro209Asp + Tyr213Met + Thr214Asn + Leu216Val Ala199Asn + Ser203Glu + Val204Met + Pro209Gly + Tyr213Leu + Leu216Ala + Gly218Asn Ala199His + Thr207Pro + Tyr213Ser + Thr214Asn + Asn217Ser + Gly218Ser + Thr219Glu Ala199Ser + Gly201Pro + Val204Gln + Thr207Ser + Thr212Asn + Tyr213Pro + Gly218Glu Ala199Ser + Pro200Asn + Tyr205Met + Tyr208Met + Thr212Asn + Thr214Gly + Leu216Met Gly201Gln + Tyr205Asn + Thr207Asn + Pro209Asp + Thr212Gly + Tyr213Val + Leu216Ile Pro200Asn + Tyr205Glu + Tyr208Met + Tyr213His + Thr214Ser + Asn217Gln + Thr219Gln Ala199Gln + Pro200Gly + Val202Met + Tyr208Ile + Thr212Asp + Leu216Met + Gly218Pro Ala199Ser + Pro200Gly + Gly201Gln + Val202Asn + Asn211Gln + Leu216Val + Gly218Pro Ala199Asn + Val202Ser + Tyr205Cys + Thr207Gly + Tyr213Leu + Thr214Asp + Asn217Ser Gly201Pro + Val202Asn + Val204Asn + Tyr205Ser + Asn211Glu + Thr212Ser + Gly218Asn Ala199Ser + Val204Cys + Thr207Ser + Asn211Ser + Thr214Gln + Ser215Glu + Asn217Gln Pro200Ser + Val202Met + Tyr205Gly + Tyr208Pro + Ser210Glu + Thr212Ser + Tyr213Gly Pro200Ser + Val202Gln + Ser203Glu + Tyr205Cys + Thr207Gln + Tyr208Ile + Asn211Ser Ala199Pro + Gly201Ser + Val204Ala + Thr207Ser + Pro209Gln + Tyr213Asp + Leu216His Gly201Ser + Ser203Asp + Val204Gln + Tyr208Thr + Asn211Gln + Leu216Ser + Thr219Ser Ala199Thr + Gly201Ser + Tyr205Pro + Tyr213Pro + Thr214Gly + Leu216Thr + Gly218Glu Pro200Asn + Gly201Gln + Tyr208Val + Thr214Gln + Ser215Asp + Gly218Ser + Thr219Asn Pro200Gln + Val202Met + Val204Gly + Tyr208Gly + Asn211Ser + Tyr213Pro + Gly218Asp Ala199Asn + Pro200Gly + Tyr205Thr + Tyr208Val + Thr212Gly + Tyr213Ser + Leu216Val Ala199Ser + Gly201Ser + Val202Gly + Tyr205Leu + Thr214Asn + Leu216Ala + Gly218Glu Ala199Ser + Gly201Gln + Val204Ser + Tyr208Asn + Pro209Ser + Thr214Asn + Ser215Glu Ala199Pro + Gly201Asn + Tyr208Cys + Tyr213Gly + Thr214Glu + Asn217Ser + Gly218Gln Ala199Pro + Val202His + Tyr208Met + Pro209Asn + Thr212Asn + Thr214Glu + Ser215Asp Ala199His + Val202Gly + Val204Ser + Thr207Gln + Pro209Asp + Ser210Asp + Asn211Ser Ala199Gly + Pro209Glu + Ser210Glu + Thr212Gln + Tyr213Ala + Leu216Pro + Thr219Asn Ala199His + Gly201Gln + Val202Met + Val204His + Thr207Gly + Pro209Glu + Ser210Asp Ala199His + Val202Gln + Thr207Gly + Thr214Gln + Leu216Thr + Asn217Asp + Gly218Glu Gly201Gln + Val204Thr + Tyr205Pro + Thr207Gly + Leu216Asn + Asn217Asp + Gly218Asp Gly201Ser + Tyr208Thr + Pro209Gln + Tyr213Ser + Asn217Asp + Gly218Glu + Thr219Gly Gly201Ser + Val202Cys + Tyr205Val + Thr207Gln + Tyr213Ile + Leu216Asp + Asn217Asp Gly201Asn + Val202Glu + Ser203Glu + Val204Gly + Thr212Gly + Leu216Val + Thr219Pro Val202Asp + Ser203Glu + Val204Asn + Thr207Pro + Tyr208Leu + Thr212Ser + Asn217Gln Gly201Gln + Tyr208Ser + Pro209Asn + Asn211Gln + Tyr213Asp + Thr214Asp + Asn217Gln Ala199Asn + Pro200Asn + Val204Thr + Ser210Asp + Asn211Glu + Tyr213Leu + Thr214Pro Gly201Ser + Val202Ala + Tyr208Met + Ser210Asp + Asn211Asp + Thr212Asp + Asn217Ser Ala199Ser + Pro200Gly + Gly201Asn + Val202Glu + Leu216Cys + Asn217Asp + Gly218Asn Ala199Asn + Pro200Ser + Ser203Asp + Tyr205Cys + Tyr208Asn + Thr214Gln + Ser215Glu Ala199Gln + Val202Gly + Ser203Glu + Tyr205Gly + Tyr213Met + Ser215Glu + Asn217Ser Gly201Asn + Val202Gly + Ser203Asp + Tyr208Ile + Tyr213Pro + Ser215Glu + Asn217Ser Val202Met + Ser203Glu + Thr207Asn + Thr214Gly + Ser215Asp + Leu216Glu + Asn217Asp Ala199Gln + Ser203Glu + Val204Cys + Asn211Ser + Thr214Gly + Asn217Glu + Thr219Pro Ala199Gln + Ser203Asp + Tyr205Thr + Thr207Ser + Thr212Asn + Asn217Asp + Gly218Asn Pro200Ser + Val202Ser + Ser203Asp + Val204Gly + Thr212Asn + Tyr213Met + Asn217Glu Ser203Glu + Tyr205Met + Tyr208Thr + Pro209Gly + Asn217Glu + Gly218Asn + Thr219Ser Ala199Gln + Tyr205Leu + Tyr208Pro + Asn211Glu + Thr212Asp + Tyr213Asp + Thr214Gly Gly201Pro + Val202Thr + Val204His + Tyr205Met + Asn211Glu + Thr212Asp + Tyr213Asp Gly201Pro + Ser203Glu + Tyr205Asp + Thr207Ser + Ser215Asp + Leu216His + Thr219Ser Ala199Asn + Val202Gln + Val204Ser + Pro209Gly + Ser210Glu + Thr212Asp + Asn217Gln Pro200Gly + Gly201Pro + Tyr208Val + Pro209Glu + Asn211Asp + Thr212Gln + Thr219Ser Ala199Asn + Pro200Gln + Val202Ala + Val204Pro + Thr207Asn + Pro209Glu + Asn211Glu Ala199Asn + Ser203Asp + Thr207Gly + Asn211Ser + Ser215Glu + Asn217Asp + Gly218Gln Pro200Gln + Ser203Glu + Val204Gly + Tyr213Pro + Ser215Glu + Asn217Glu + Thr219Gln Pro200Gly + Val202Asp + Ser203Asp + Thr207Ser + Thr212Gly + Ser215Glu + Gly218Ser Val202Glu + Ser203Asp + Tyr208Ser + Asn211Gln + Tyr213Ala + Ser215Asp + Thr219Ser Ala199Gln + Ser203Asp + Tyr205Met + Thr214Asp + Ser215Glu + Leu216Cys + Asn217Gln Gly201Gln + Ser203Glu + Tyr205Glu + Pro209Gln + Asn211Ser + Thr214Glu + Leu216Glu Gly201Asn + Val202Cys + Ser203Asp + Tyr205Asp + Thr214Asp + Leu216Gly + Thr219Pro Ala199Gly + Val202Met + Ser203Glu + Val204Pro + Tyr205Glu + Thr207Ser + Thr214Asp Gly201Ser + Val204Met + Pro209Gly + Asn211Ser + Tyr213Cys + Thr214Glu + Leu216Asp Gly201Pro + Val202Asp + Thr207Gln + Thr212Ser + Asn217Asp + Gly218Asn + Thr219Asp Pro200Ser + Val204Gly + Tyr205Gly + Tyr208Cys + Thr214Ser + Ser215Glu + Asn217Glu Gly201Asn + Tyr205Ile + Tyr213Ile + Thr214Asn + Ser215Glu + Asn217Asp + Thr219Pro Pro200Ser + Val202Gln + Tyr208His + Thr212Pro + Thr214Ser + Ser215Glu + Asn217Glu Pro200Gly + Val204Gln + Thr207Ser + Tyr213Leu + Thr214Gln + Ser215Glu + Asn217Glu Thr207Gly + Ser210Asp + Asn211Asp + Thr212Gly + Tyr213Asp + Asn217Gln + Thr219Gln Ser203Glu + Thr207Asn + Tyr208His + Asn211Gln + Thr214Asp + Leu216Glu + Asn217Glu Gly201Pro + Val202Met + Val204Ser + Tyr205His + Thr212Gly + Asn217Glu + Thr219Asp Ala199Asn + Val204Cys + Tyr208Cys + Tyr213Ala + Thr214Glu + Leu216Asp + Asn217Glu Pro200Gln + Thr207Gly + Tyr208Cys + Thr214Asp + Ser215Asp + Leu216Gln + Asn217Asp Ala199Gln + Val204His + Asn211Ser + Thr212Ser + Thr214Glu + Ser215Glu + Asn217Glu Val202Asp + Ser203Asp + Tyr205Ala + Thr207Gln + Tyr213Met + Thr214Glu + Ser215Asp Ala199Gly + Pro200Gln + Val204Cys + Thr212Glu + Thr214Glu + Ser215Glu + Leu216Gln Val204Ala + Thr207Gly + Ser210Asp + Asn211Asp + Thr212Glu + Thr214Glu + Asn217Ser Gly201Pro + Pro209Ser + Ser215Glu + Leu216Glu + Asn217Asp + Gly218Ser + Thr219Asp Pro200Asn + Gly201Pro + Ser203Asp + Thr207Gln + Asn217Asp + Gly218Ser + Thr219Glu Ala199Pro + Val202Gly + Ser203Asp + Pro209Gln + Thr212Asn + Ser215Glu + Gly218Asp Ala199Gln + Pro200Ser + Gly201Ser + Tyr205Gln + Ser210Asp + Thr212Glu + Thr214Asp Ala199Pro + Pro200Ser + Thr207Gln + Ser210Glu + Thr212Asp + Thr214Asp + Thr219Ser Ala199His + Ser203Asp + Val204Gly + Thr207Gly + Asn211Gln + Thr214Ser + Gly218Glu Gly201Asn + Val202Asp + Val204Gly + Tyr208Val + Asn211Gln + Asn217Ser + Thr219Asp Val202Asn + Tyr205Glu + Thr207Gln + Tyr208Leu + Thr214Asp + Asn217Asp + Gly218Pro Ala199Asn + Gly201Asn + Ser203Asp + Tyr208Asn + Thr214Glu + Leu216Cys + Thr219Gly Val202Cys + Ser203Asp + Val204Ser + Tyr208Thr + Thr214Asp + Asn217Gln + Thr219Pro Ser203Asp + Tyr208Pro + Pro209Ser + Asn211Ser + Tyr213Met + Thr214Asp + Thr219Pro Ala199Asn + Pro200Gln + Ser203Asp + Thr212Glu + Thr214Asp + Ser215Glu + Asn217Ser Pro200Asn + Gly201Asn + Val202Glu + Ser203Asp + Thr212Gly + Ser215Glu + Thr219Asp Ala199Gln + Gly201Gln + Pro209Gly + Ser210Glu + Thr212Pro + Tyr213Glu + Leu216Ala Pro200Ser + Val204Ser + Thr207Asn + Tyr208Leu + Ser210Asp + Asn211Ser + Tyr213Asp Ala199His + Ser203Asp + Tyr205Asp + Thr207Asn + Tyr208Ile + Leu216Ser + Gly218Asp Ala199Gln + Gly201Pro + Val202Glu + Tyr205Glu + Pro209Gly + Thr214Asp + Leu216Gln Gly201Asn + Val204Pro + Tyr208Cys + Ser210Glu + Asn211Glu + Thr212Pro + Thr214Asp Ala199His + Pro200Asn + Tyr208Val + Thr214Glu + Ser215Glu + Leu216Glu + Thr219Asp Pro200Gly + Val202Glu + Tyr205Pro + Tyr208Ala + Thr214Glu + Asn217Asp + Gly218Asn Val202Glu + Tyr205His + Pro209Gln + Thr214Glu + Ser215Asp + Asn217Ser + Gly218Asp Pro200Ser + Tyr205Leu + Thr212Gln + Thr214Asp + Ser215Glu + Gly218Glu + Thr219Asn Ser203Asp + Val204Gly + Tyr205Ser + Thr212Glu + Thr214Pro + Ser215Asp + Asn217Glu Pro200Asn + Val204Gln + Pro209Glu + Ser210Glu + Asn211Glu + Thr212Asn + Ser215Asp Ala199Gln + Thr207Pro + Tyr208Cys + Pro209Gln + Tyr213Asp + Thr214Asn + Leu216Asp Ala199Gly + Pro209Glu + Ser210Asp + Thr214Glu + Ser215Glu + Gly218Pro + Thr219Gly Ala199His + Thr207Ser + Tyr208Thr + Pro209Gly + Ser215Asp + Asn217Gln + Gly218Glu Ala199Ser + Ser203Glu + Tyr208Ala + Asn211Gln + Ser215Glu + Leu216Cys + Thr219Asp Gly201Pro + Val202Gly + Ser203Glu + Tyr205Thr + Tyr208Ala + Ser215Asp + Thr219Asp Pro200Gln + Val202His + Ser203Glu + Asn211Gln + Thr212Asp + Thr214Asp + Thr219Gly Gly201Pro + Ser203Asp + Thr207Gln + Ser210Glu + Thr214Glu + Ser215Asp + Thr219Ser Ala199Ser + Tyr205Gly + Thr207Ser + Ser210Asp + Tyr213Glu + Ser215Glu + Leu216Glu Val204Thr + Thr207Pro + Ser210Glu + Asn211Glu + Thr212Glu + Gly218Asp + Thr219Ser Val202Asp + Ser203Asp + Thr207Gly + Pro209Asn + Asn211Glu + Asn217Asp + Gly218Ser Pro200Ser + Ser203Asp + Val204Pro + Tyr208Ile + Thr214Asp + Asn217Ser + Gly218Asp Pro200Ser + Gly201Pro + Val202Gly + Ser203Glu + Tyr205Glu + Pro209Asp + Tyr213Glu Ala199Gly + Gly201Gln + Val202Ser + Asn211Asp + Ser215Glu + Leu216Asp + Asn217Glu Ala199Thr + Tyr205Gln + Thr207Gln + Pro209Gln + Thr212Asp + Ser215Glu + Gly218Gln Ala199Gln + Val204Ser + Tyr205Glu + Thr207Asn + Ser210Glu + Thr212Glu + Asn217Gln Tyr205Val + Tyr208Pro + Pro209Gln + Ser210Asp + Thr212Glu + Ser215Glu + Leu216Gly Ala199Pro + Gly201Ser + Thr214Glu + Ser215Glu + Asn217Gln + Gly218Glu + Thr219Asp Ser203Glu + Ser210Asp + Asn211Gln + Thr212Glu + Tyr213Ser + Thr214Glu + Leu216Cys Ala199His + Pro200Gln + Pro209Asn + Tyr213Gln + Thr214Asp + Leu216Glu + Thr219Asp Ala199Asn + Ser203Asp + Tyr205Glu + Thr207Asn + Pro209Asn + Asn211Glu + Leu216Glu Ala199Thr + Gly201Pro + Ser210Glu + Thr214Asp + Ser215Asp + Asn217Ser + Thr219Ser Ser203Glu + Thr207Pro + Tyr208Ala + Ser210Asp + Ser215Glu + Asn217Asp + Thr219Pro Val202Asp + Val204Cys + Tyr205Asp + Tyr208Ala + Pro209Gly + Gly218Gln + Thr219Glu Val202Ala + Val204Thr + Tyr208Leu + Asn211Glu + Thr212Glu + Ser215Asp + Leu216Glu Pro200Asn + Val202Gln + Thr207Gln + Pro209Ser + Asn211Asp + Thr214Glu + Leu216Asp Ala199Asn + Val202Met + Thr207Asn + Pro209Glu + Tyr213Glu + Ser215Glu + Asn217Asp Ala199Gly + Val202Cys + Val204Gln + Tyr205Asp + Ser210Asp + Asn211Gln + Thr214Glu Ala199His + Tyr208Thr + Pro209Asp + Ser215Asp + Leu216Met + Asn217Asp + Gly218Asp Ala199Asn + Pro200Asn + Gly201Gln + Pro209Asp + Thr214Ser + Ser215Asp + Leu216Asp Ala199Asn + Pro200Gln + Gly201Ser + Val202Asp + Asn211Asp + Ser215Glu + Leu216Asp Ala199Thr + Val202Asp + Val204Cys + Ser210Asp + Tyr213Ile + Ser215Asp + Leu216Asp Gly201Ser + Val202Glu + Tyr205Gly + Tyr208Leu + Ser210Asp + Ser215Glu + Asn217Asp Gly201Gln + Thr207Ser + Tyr208His + Pro209Glu + Asn211Glu + Tyr213Asp + Asn217Glu Pro200Gly + Gly201Asn + Ser203Glu + Tyr208Cys + Thr212Asn + Asn217Gln + Thr219Glu Ala199Gly + Gly201Asn + Val202His + Val204Ala + Pro209Asp + Ser210Glu + Ser215Glu Gly201Asn + Val202Glu + Pro209Gly + Ser210Glu + Thr212Glu + Tyr213Thr + Thr214Glu Val202Cys + Val204Ala + Tyr205Glu + Thr212Ser+ Ser215Glu + Asn217Gln + Thr219Asp Gly201Ser + Ser203Glu + Ser210Glu + Asn211Ser + Thr212Glu + Ser215Glu + Leu216Pro Ala199His + Ser203Glu + Thr207Gln + Ser210Asp + Thr212Asp + Ser215Asp + Gly218Gln Ala199Gln + Pro200Asn + Gly201Gln + Val204Asn + Thr214Asp + Ser215Glu + Thr219Glu Ala199Pro + Gly201Pro + Ser203Asp + Tyr205Val + Pro209Glu + Ser215Glu + Thr219Pro Pro200Gly + Ser203Asp + Thr207Gln + Asn211Glu + Tyr213Glu + Leu216Thr + Asn217Gln Val202Asp + Tyr205Glu + Tyr208Gly + Thr212Asp + Tyr213Ala + Leu216Ile + Thr219Pro Pro200Ser + Val202Glu + Tyr205Asp + Asn211Glu + Thr212Asp + Tyr213Cys + Thr219Gly Val204Pro + Tyr205Asp + Asn211Asp + Thr214Pro + Ser215Asp + Asn217Ser + Thr219Pro Gly201Asn + Ser203Asp + Pro209Gln + Tyr213Thr + Thr214Asp + Gly218Asn + Thr219Glu Pro200Gln + Val204Thr + Thr212Asn + Tyr213Glu + Thr214Pro + Asn217Asp + Gly218Asp Ala199Gln + Gly201Gln + Val204Asn + Tyr208Met + Ser210Glu + Thr214Glu + Asn217Ser Gly201Pro + Val204Thr + Tyr208Gly + Asn211Asp + Thr214Asp + Asn217Glu + Gly218Asp Pro200Asn + Pro209Glu + Ser210Asp + Asn211Ser + Thr214Ser + Leu216Cys + Thr219Asp Ser203Asp + Thr207Ser + Tyr208Asn + Pro209Asp + Ser210Asp + Thr214Gln + Asn217Gln Val204Ser + Tyr208Pro + Pro209Ser + Asn211Asp + Leu216Met + Asn217Asp + Gly218Asp Tyr205Ser + Tyr208Cys + Pro209Asn + Asn211Asp + Thr212Asn + Leu216Glu + Asn217Asp Pro200Gly + Ser210Asp + Thr212Pro + Tyr213Val + Asn217Ser + Gly218Asp + Thr219Asp Val202Cys + Tyr205Ala + Thr207Asn + Asn211Asp + Tyr213Val + Ser215Asp + Leu216Asp Ala199Gln + Val202Glu + Ser203Asp + Val204Thr + Pro209Gln + Asn211Asp + Tyr213Pro Gly201Pro + Val202Glu + Ser203Glu + Val204Gly + Pro209Gln + Thr212Glu + Leu216Ala Pro200Gln + Val202Asp + Ser203Asp + Tyr208Ala + Thr212Glu + Leu216Val + Gly218Gln Ala199Thr + Gly201Gln + Val202Gly + Ser210Glu + Asn211Asp + Ser215Asp + Leu216Ala Ala199Gly + Gly201Gln + Val204Asn + Ser210Asp + Asn211Asp + Ser215Glu + Thr219Asn Ala199Asn + Val202Ala + Ser203Asp + Val204Gln + Ser210Glu + Thr212Glu + Leu216Glu Val202Asp + Val204Asn + Pro209Ser + Ser210Asp + Asn211Asp + Tyr213Asn + Leu216Met Val202Pro + Ser203Asp + Val204Ser + Thr212Glu + Tyr213Asp + Thr214Asn + Gly218Asp Ser203Glu + Val204Pro + Tyr205Asp + Pro209Glu + Thr212Gln + Tyr213Asn + Gly218Asp Pro200Ser + Thr207Gly + Ser210Glu + Thr212Gly + Thr214Glu + Ser215Asp + Gly218Glu Val202Met + Val204His + Ser210Glu + Thr212Asp + Tyr213Ile + Leu216Asp + Gly218Glu Ser203Glu + Tyr208Ser + Asn211Asp + Thr212Pro + Thr214Asn + Ser215Glu + Gly218Pro Ala199Thr + Gly201Pro + Ser203Asp + Tyr205Gly + Asn211Asp + Ser215Glu + Asn217Ser Pro200Gln + Gly201Asn + Ser203Glu + Pro209Asn + Asn211Asp + Thr212Gly + Ser215Glu

TABLE 29 Multi-loop Double Mutation Variants Leu 95Gly + Ser203Glu Thr 58Gln + Asp 59Glu Tyr103Cys + Leu216Gln Val202Gln + Thr212Gly Gly126Gln + Gly156Glu Thr214Gly + Ser215Glu Asn 96Gln + Ile106Ala Gly101Asp + Tyr205Pro Asn 61Ser + Gly165Gln Gly 64Gln + Thr132Gly Asp 59Glu + Tyr205Asn Ser100Glu + Tyr205His Gln161Glu + Tyr205Pro Thr 58Asp + Tyr205Ile Asn 61Asp + Asn162Ser Gln161Glu + Pro209Gln Gly 62Asp + Pro209Asn Ile106Leu + Asn211Asp Tyr166Pro + Ser215Glu Ile106Leu + Pro209Glu Thr163Pro + Val202Cys Thr 65Pro + Leu216Ser Thr132Gly + Phe188Val Gly156Glu + Ala186Thr Pro128Ser + Ser160Asp Gly 62Gln + Gly165Asp Leu 95Gln + Pro128Glu Ser 97Glu + Asn211Ser Leu 95Asn + Phe188Leu Leu125Gln + Gly159Glu Ser187Glu + Tyr213Ala Tyr205Cys + Asn211Asp Asn162Gln + Tyr213Pro Ser158Asp + Leu216Gln Gly156Asp + Val202Ala Ser100Asp + Tyr205Ile Gly 99Asn + Thr214Glu Thr 58Asp + Gly130Gln Gly156Glu + Tyr208Pro Ala105Pro + Leu216Ile Ser187Asp + Tyr205Gln Leu125Asn + Ser187Glu Thr 65Gln + Thr102Glu Tyr103Ile + Tyr205Glu Thr132Asp + Gly218Gln Asn211Ser + Thr212Asp Thr212Gln + Ser215Glu Thr132Pro + Tyr205Gly Tyr103Glu + Thr132Ser Gly165Gln + Ser203Glu Gly 60Pro + Thr 65Pro Gly 99Asn + Gly153Asn Pro128Asn + Val202His A1a105Glu + Gly130Ser Thr212Gly + Gly218Glu Phe188Val + Tyr205Ser Asn217Glu + Thr219Gly Leu 95Gln + Ser215Asp Val 94Gln + Thr212Glu Asn 61Ser + Ser203Asp Ser129Glu + Asn154Gln Gly 62Glu + Thr219Asn Ser 97Glu + Tyr213His Gly 99Asp + Ile164Thr Gly101Pro + Ser215Asp Thr 58Pro + Tyr205Gln Gly101Gln + Tyr208Gln Asn 61Gln + Thr212Asp Thr212Asp + Leu216Ile Asn211Glu + Leu216His Gly153Glu + Thr163Gln Thr132Asn + Tyr205Asp Asn 61Ser + Ser203Glu Ser215Asp + Leu216Ser Gly 60Asp + Met198Gln Ser158Glu + Tyr205Met Phe188Leu + Thr212Glu Gly130Pro + Ser190Glu Gly101Asp + Gly126Ser Ala105Gly + Ser157Asp Asn 61Ser + Ser131Asp Asn 61Ser + Phe188Ser Ser100Glu + Asn162Gln Thr 58Gly + Pro128Asp Ser100Glu + Tyr205Ala Gly127Glu + Tyr213Thr Asn154Glu + Leu216Gln Ser 97Asp + Ala199Pro Ser203Glu + Leu216Ser Gly126Gln + Ser215Glu Gly 60Ser + Asn154Glu Thr 65Ser + Gly156Asp Pro128Ser + Thr163Gln Ser215Asp + Leu216Val Ser129Glu + Leu216Ala Asn211Asp + Tyr213Leu Val 94Ser + Phe188Val Ser158Glu + Thr212Gln Gly 99Pro + Asn154Gln Val202Ser + Tyr205Glu Tyr205Val + Asn217Glu Val 94Gly + Ser215Asp Tyr205Glu + Tyr213Pro Asn 96Ser + Gly127Glu Tyr205Thr + Thr214Gly Gly 62Ser + Thr 65Ser Thr 65Glu + Pro128Asn Ser215Asp + Leu216Gln Asp 59Glu + Gly 60Pro Val 94Thr + Leu216Met Ser 97Glu + Thr212Gln Gly101Asp + Val202Asn Ser129Asp + Val202Gly Tyr103Ala + Thr132Ser Leu 95Met + Leu216Asp Ser100Asp + Gly101Pro Ser100Asp + Thr219Pro Val 94Asn + Ser215Asp Gly153Asn + Tyr205Asp Thr 58Glu + Ile106Cys Thr 65Glu + Tyr103Pro Val 94Met + Ser 98Glu Ser210Glu + Gly218Ser Leu125Pro + Ala199His Ile106Gly + Thr212Glu Tyr205Ile + Thr214Pro Gly130Glu + Tyr208His Ser187Asp + Leu216Val Thr102Glu + Thr219Gln Leu125Ser + Gly153Glu Gly153Asn + Val202Asp Thr163Glu + Tyr205Gly Ser129Glu + Tyr166Pro Gly153Gln + Pro209Gln Thr212Ser + Ser215Glu Thr163Asp + Tyr205His Asn154Glu + Tyr166Thr Asn 96Ser + Ala105Gly Gly101Ser + Phe188Gly Gly153Glu + Gly218Asn Ser100Asp + Thr212Pro Gly126Pro + Gly156Asp Leu125Ser + Ser215Glu Asp 59Glu + Thr 65Gly Gly156Pro + Gly159Asp Val202Cys + Tyr213Ser Leu 95Met + Ser131Glu Ser 97Asp + Gly 99Ser Asn 61Gln + Leu 95Asp Gly126Asn + Tyr205Glu Glyl59Pro + Gly218Asn Leu 95Thr + Leu216Ala Ala105Glu + Leu216Thr Thr102Glu + Gly130Pro Gly 64Gln + Ser215Glu Tyr103Asp + Gly126Asn Gly101Glu + Gly127Gln Thr132Asn + Ser160Asp Thr 58Pro + Gly153Asn Ser203Glu + Thr214Ser Tyr103His + Ser129Asp Ser 98Asp + Pro209Ser Asp 59Glu + Tyr205Ser Gly 60Gln + Ser215Asp Thr132Asp + Val202Asn Gly218Ser + Thr219Gly Ser190Asp + Val202Thr Asp 59Glu + Gln161Asn Leu216Asn + Asn217Asp Val 94Asp + Tyr205Asn Tyr103Ser + Ser203Asp Gly 99Pro + Phe188Gln Ser 97Asp + Leu216His Thr163Asp + Leu216Asn Asn 61Asp + Thr132Ser Leu 95Asn + Ser215Asp Ser100Asp + Thr132Gly Tyr166Thr + Asn211Gln Thr207Pro + Leu216Thr Ile106Ala + Gly165Asp Tyr205Asn + Ser215Glu Leu 95Asn + Val202Gln Ser160Asp + Phe188Gly Gly 64Ser + Asn 96Gln Gly101Asp + Asn154Gln Gly 64Asn + Ser157Glu Phe 88Asp + Tyr213Asn Ser215Glu + Leu216Ile Tyr103His + Ser187Glu Asn 96Ser + Thr214Asn Gly 62Asn + Ser 98Asp Ile106Ser + Ser157Glu Thr 65Glu + Thr212Gly Gly127Ser + Ser215Asp Gly156Ser + Asn217Glu Ser 98Asp + Tyr103Thr Gly127Asn + Gly165Pro Gly153Glu + Leu216Met Ser129Asp + Gly165Pro Asn 61Asp + Thr163Gln Gly126Asn + Tyr205Met Gly165Gln + Ser215Glu Thr 65Glu + Gly101Pro Val 94Gln + Gly165Glu Tyr103Ile + Ser190Glu Asn217Gln + Gly218Ser Ser 98Glu + Gly 99Asn Thr102Asp + Thr212Ser Thr212Ser + Ser215Asp Gly130Glu + Pro209Asn Pro128Glu + Gly165Pro Thr 58Asn + Leu125Glu Leu125His + Thr163Ser Thr212Asp + Tyr213Ala Thr 65Pro + Ser210Glu Val 94Thr + Thr132Gly Tyr205cys + Thr214Gln Gly127Gln + Ala186Gln Gly127Glu + Tyr205cys Ser 98Glu + Gly130Ser Gly127Glu + Phe188Tyr Phe188Asp + Asn211Ser Gly 64Asn + Leu216Asp Ser203Glu + Thr212Ser Tyr166Val + Ser210Asp Gly165Glu + Tyr205Met Asn 96Gln + Thr132Glu Ser 98Glu + Gly101Pro Leu125Cys + Ser215Asp Leu125Cys + Gly126Ser Gly 99Gln + Ser210Asp Ser160Asp + Phe188Leu Val204Gln + Ser215Glu Asn 61Gln + Leu216Asp Leu125Gly + Ser155Asp Ala186Pro + Val204Gln Ile106Met + Ser160Asp Leu125Ile + Leu216Ser Gly 60Ser + Ser 97Glu Gly165Asp + Tyr213Gln Pro128Glu + Asn154Gln Gly 60Asp + Gly126Pro Ser203Glu + Leu216Ala Gly126Glu + Val202Gly Thr132Asp + Tyr213Ser Ser157Asp + Thr212Ser Gly130Ser + Ser203Glu Gly 99Asn + Gly130Ser Asp 59Glu + Gly159Asn Gly153Asn + Thr212Asp Glyl53Asp + Tyr205Ser Ser100Glu + Tyr205Thr Gly126Asn + Asn154Glu Ser190Asp + Tyr205Val Thr102Glu + Tyr166Thr Ser157Glu + Pro209Gly Ser100Asp + Tyr205Leu Ser131Glu + Thr132Asn Gly127Asp + Leu216Thr Ser210Glu + Asn217Gln Asn 61Asp + Ile106Gln Ala105Pro + Tyr205Pro Ser190Asp + Leu216Cys Ser203Glu + Tyr205Thr Leu 95Asp + Asn 96Ser Tyr205Ala + Thr212Glu Thr163Gln + Phe188Glu Tyr103Ala + Thr212Asp Ser155Glu + Gly218Asn Val 94Thr + Thr163Ser Thr102Glu + Asn217Gln Gly159Ser + Val202Glu Asn154Ser + Tyr166Ala Asp 59Glu + Phe188Gly Thr163Gln + Gly218Ser Gly153Asn + Tyr205Val Ser129Glu + Leu216Pro Gly126Ser + Thr212Glu Ser203Asp + Leu216Asn Gly126Glu + Thr212Pro Val 94Pro + Ala186Glu Ser129Asp + Gly153Asn Gly165Ser + Ser215Asp Gly 60Gln + Ser187Glu Ser203Asp + Leu216His Gly101Glu + Ile106Met Val 94Glu + Gly127Gln Val202Gly + Leu216His Asp 59Glu + Thr132Asn Val202Asn + Tyr205Pro Leu 95Ile + Ser215Glu Pro128Glu + Leu216Pro Ser 97Asp + Tyr213Met Ser187Glu + Leu216cys Asn154Ser + Gly156Gln Leu125Gly + Gly126Ser Asn154Ser + Leu216Asn Gly126Asn + Val202Asn Asn 96Gln + Gly127Ser Gly 64Ser + Thr212Gly Ser160Asp + Asn211Ser Ser210Glu + Gly218Gln Tyr205Ala + Thr212Asp Ser158Asp + Leu216Ser Thr212Gly + Ser215Glu Ala199Ser + Val202Ala Ser129Glu + Tyr205Asn Asn154Gln + Gly201Gln Ser190Glu + Thr212Ser Thr 58Pro + Ser160Asp Gly127Glu + Gly130Ser Asn154Glu + Thr212Gly Gly153Ser + Gln161Asn Tyr205Thr + Ser210Glu Leu125Gly + Pro200Gln Tyr103Val + Thr132Gly Gly126Asp + Leu216Ala Ser100Glu + Gly127Asn Ser129Asp + Ala186Ser Ser160Glu + Tyr205Ser Thr163Glu + Ile164His Gly159Ser + Gln161Glu Asp 59Glu + Gly159Pro Gly101Asp + Tyr166Cys Thr 58Asn + Ser129Glu Asn 6lGlu + Thr214Ser Gly127Ser + Ser203Glu Gly127Pro + Gly130Asp Gly127Glu + Val202Gly Phe188Met + Ser203Glu Ser 97Glu + Tyr166Asn Gly 60Glu + Gly 99Asn Asn162Glu + Asn217Ser Thr102Asp + Leu216Gly Gly 99Gln + Ala105Thr Gly153Ser + Ser203Glu Tyr103Pro + Ser158Glu Ser187Glu + Tyr205Pro Ser160Asp + Val204Thr Ser100Asp + Leu216His Gly101Asn + Ser203Asp Asn 96Ser + Tyr205Pro Thr132Gly + Thr212Pro Ile106Pro + Val202Cys Gly 62Ser + Pro209Glu Phe188Cys + Leu216Asp Leu 95Cys + Ser104Glu Ser104Asp + Tyr205Met Tyr205Glu + Thr214Gly Gly153Glu + Leu216Asn Val 94Thr + Ser100Glu Thr 65Asp + Val202His Val202Gln + Ser215Asp Val 94Ser + Ala105Glu Thr 58Pro + Gly153Asp Ala105Asn + Ser203Glu Ser104Asp + Ala105Gly Gly126Gln + Ile164Val Gly153Ser + Ser215Glu Pro200Gly + Ser215Glu

TABLE 33 Multi-loop Triple Mutation Variants Thr58Gln + Leu95Gly + Ser203Glu Asp59Glu + Tyr103Cys + Leu216Gln Gly156Glu + Val202Gln + Thr212Gly Gly126Gln + Thr214Gly + Ser215Glu Asn96Gln + Ile106Ala + Gly156Glu Asn61Ser + Gly101Asp + Tyr205Pro Gly64Gln + Thr132Gly + Gly165Gln Thr65Pro + Thr163Pro + Val202Cys Thr132Gly + Phe188Val + Leu216Ser Leu95Asn + Ser97Glu + Asn211Ser Asn162Gln + Ser187Asp + Tyr213Pro Ser100Asp + Val202Ala + Tyr205Ile Thr58Asp + Gly99Asn + Gly130Gln Gly156Glu + Tyr208Pro + Leu216Ile Thr65Gln + Leu125Asn + Ser187Glu Asn211Ser + Thr212Asp + Gly218Gln Tyr205Gly + Thr212Gln + Ser215Glu Thr58Glu + Gly60Pro + Thr65Pro Asn96Gln + Gly153Pro + Leu216Val Gly99Asn + Gly153Asn + Pro209Glu Ala105Glu + Pro128Asn + Val202His Tyr205Ser + Thr212Gly + Gly218Glu Gly99Gln + Asn217Glu + Thr219Gly Leu95Gln + Val202Thr + Ser215Asp Gly101Pro + Ile164Thr + Ser215Asp Thr58Pro + Gly130Asp + Tyr205Gln Gly101Gln + Ser190Glu + Tyr208Gln Asn61Gln + Thr212Asp + Leu216Ile Asn61Ser + Thr132Asn + Tyr205Asp Gly130Pro + Phe188Leu + Thr212Glu Asn61Ser + Ala105Gly + Ser131Asp Asn61Ser + Asn162Gln + Phe188Ser Gly126Gln + Ser203Glu + Leu216Ser Thr65Ser + Gly156Asp + Thr163Gln Pro128Ser + Ser215Asp + Leu216Val Val94Ser + Ser100Asp + Phe188Val Gly99Pro + Asn154Gln + Ser203Glu Tyr166Asp + Tyr205Thr + Thr214Gly Gly62Ser + Thr65Ser + Asn211Glu Thr65Glu + Pro128Asn + Leu216Gln Val94Thr + Thr212Gln + Leu216Met Ser129Asp + Thr132Ser + Val202Gly Leu95Met + Tyr103Ala + Leu216Asp Val94Met + Ser98Glu + Tyr103Pro Leu125Pro + Ala199His + Ser203Glu Ile106Gly + Tyr205Ile + Thr214Pro Ser129Glu + Tyr166Pro + Tyr205Gly Pro209Gln + Thr212Ser + Ser215Glu Asn154Glu + Tyr166Thr + Tyr205His Asn96Ser + Gly101Ser + Ala105Gly Gly153Glu + Phe188Gly + Gly218Asn Gly126Pro + Gly156Asp + Thr212Pro Gly156Pro + Gly159Asp + Val202Cys Leu95Met + Ser97Asp + Gly99Ser Gly126Asn + Gly159Pro + Gly218Asn Thr65Gln + Leu95Thr + Leu216Ala Tyr103Asp + Gly126Asn + Gly127Gln Thr58Pro + Ala105Glu + Gly153Asn Gly60Gln + Tyr205Ser + Ser215Asp Thr132Asp + Gly218Ser + Thr219Gly Tyr103Ser + Phe188Gln + Ser203Asp Ser97Asp + Gly99Pro + Leu216His Asn61Asp + Thr132Ser + Leu216Asn Leu95Asn + Thr132Gly + Ser215Asp Tyr166Thr + Asn211Gln + Leu216Thr Ile106Ala + Gly165Asp + Thr207Pro Leu95Asn + Val202Gln + Ser215Glu Gly64Ser + Gly101Asp + Asn154Gln Thr58Asn + Gly64Asn + Ser157Glu Gly62Asn + Asn96Ser + Thr214Asn Gly130Asp + Phe188Asn + Gly218Pro Gly99Ser + Pro128Glu + Leu216Gly Val94Ala + Pro209Ser + Ser215Asp Leu95Thr + Gly126Gln + Leu216Gly Asp59Glu + Gly159Ser + Tyr205Pro Val202Gln + Ser203Glu + Tyr205Asn Thr58Ser + Ser190Glu + Tyr205Pro Leu95Asn + Tyr205Ala + Pro209Glu Thr58Asn + Gly153Pro + Ser210Glu Ser131Glu + Thr132Asn + Gly165Asn Gly127Ser + Thr212Asn + Ser215Asp Thr65Gly + Thr102Asp + Gly127Asn Gly60Asn + Tyr205Thr + Asn217Gln Ala199Ser + Val202Ala + Thr212Gly Thr58Pro + Gly130Ser + Ser160Asp Gly153Ser + Asn154Glu + Thr212Gly Leu125Gly + Tyr205Thr + Ser210Glu Tyr103Val + Thr132Gly + Pro200Gln Ser100Glu + Gly127Asn + Leu216Ala Ser160Glu + Ala186Ser + Tyr205Ser Gly159Ser + Thr163Glu + Ile164His Phe188Met + Val202Gly + Ser203Glu Thr102Asp + Ala105Thr + Leu216Gly Thr58Asp + Asn96Ser + Tyr205Pro Ser97Asp + Thr132Gly + Thr212Pro Ile106Pro + Val202Cys + Ser215Asp Leu95Cys + Phe188Cys + Leu216Asp Thr65Asp + Val94Thr + Val202His Thr58Pro + Ala105Asn + Gly153Asp Ser104Asp + Ala105Gly + Ile164Val Gly126Gln + Ser203Asp + Leu216His Ser160Asp + Gly218Gln + Thr219Asn Gly62Ser + Ala105Glu + Pro128Ser Thr58Gly + Ile106Gly + Leu216Asp Ser97Asp + Thr163Gln + Ala186Ser Thr65Glu + Gly126Gln + Leu216His Phe188Ile + Ser203Glu + Leu216Thr Thr132Gln + Gly165Asp + Thr212Ser Ser129Asp + Asn211Ser + Leu216Thr Leu125Ile + Asn154Ser + Leu216Met Thr65Pro + Gly101Pro + Thr212Asp Thr102Asn + Thr212Gly + Leu216Asp Thr65Gln + Tyr205Gln + Leu216Met Leu125Asn + Pro209Gly + Thr214Glu Leu125Pro + Val202His + Ser215Asp Ser160Glu + Gln161Asn + Tyr205Val Gly126Ser + Thr212Glu + Gly218Asn Val94Thr + Ser155Asp + Thr207Gly Ser157Glu + Tyr208Gln + Thr212Gln Tyr205Met + Thr212Gln + Ser215Glu Pro128Gln + Thr132Pro + Tyr213Asn Gly60Ser + Gly156Asp + Pro209Asn Asn162Asp + Thr163Asn + Ala199Gly Gly165Ser + Tyr205Thr + Ser215Glu Ser100Asp + Gly156Pro + Tyr205Gln Ala105His + Ser157Glu + Ala186Asn Asn162Gln + Ser190Glu + Val202Ser Gly99Gln + Gly126Gln + Gln161Asp Gly126Asp + Gly159Ser + Thr212Gly Gly62Glu + Asn154Gln + Leu216Gln Asn154Asp + Tyr205Ser + Tyr213Met Asn61Asp + Leu95Ser + Val204Gln Thr102Gln + Gly159Gln + Ser190Glu Pro128Asn + Gly159Asn + Asn211Ser Gly159Ser + Thr163Gly + Ala199Thr Asn211Gln + Tyr213Gln + Ser215Glu Gly99Ser + Leu125Gly + Ser215Glu Thr65Gly + Ser190Glu + Thr219Ser Val94Met + Asn162Ser + Leu216Met Leu95Ser + Tyr103His + Gly156Glu Thr58Gly + Asn211Glu + Leu216Ala Ala105Gly + Thr212Pro + Gly218Glu Ser158Asp + Gly159Ser + Tyr205Pro Thr58Gln + Ser158Glu + Thr163Gly Thr65Pro + Leu125Met + Thr163Gly Gly62Asn + Gly126Pro + Ser210Glu Leu95Met + Thr132Asp + Asn211Ser Tyr103Ile + Gly159Asp + Val202Gly Gly101Gln + Tyr166Pro + Asn211Gln Pro128Gly + Tyr132Pro + Thr214Gln Pro200Ser + Thr212Asp + Leu216Gly Thr212Asp + Leu216Met + Gly218Gln Thr163Gln + Tyr205Cys + Thr212Pro Thr58Gln + Ser98Glu + Thr212Pro Thr102Pro + Ser155Asp + Tyr205Asn Gly62Ser + Gly99Glu + Val202Ser Leu95Asp + Ile106Gln + Gly126Asn Ser100Glu + Gly101Gln + Val202Ala Thr58Ser + Ser100Glu + Ile164Leu Gly101Ser + Phe188Asp + Tyr213Gly Ser104Glu + Gly156Gln + Thr212Gly Thr65Asp + Val94Gly + Tyr205Met Ser203Glu + Thr212Ser + Tyr213Val Thr65Ser + Leu125Glu + Gln161Ser Thr65Gly + Tyr205Asp + Tyr213Thr Gly101Pro + Ser104Asp + Thr163Gln Ala105Gly + Asn154Asp + Pro209Gly Gly126Gln + Gln161Ser + Leu216Ala Gly153Gln + Tyr166Cys + Gly218Asp Gly99Gln + Val204Pro + Thr219Glu Thr58Ser + Ser97Asp + Thr212Pro Asn162Ser + Met198Gly + Val202Thr Val94Ser + Leu125Asp + Tyr205Thr Ser157Asp + Tyr205Ser + Leu216Thr Gly165Asn + Ser215Glu + Leu216Pro Gly156Asn + Thr163Glu + Ala199Ser Pro128Ser + Gly156Glu + Thr219Gly Asn96Gln + Gly126Gln + Ser203Glu Gly159Asp + Gly165Pro + Tyr213Ile Thr58Gln + Gly153Ser + Asn217Gln Pro128Ser + Thr132Glu + Thr212Gly Gly156Gln + Tyr205Pro + Ser215Glu Gly62Ser + Tyr205Ile + Leu216Asp Leu125Pro + Gly159Glu + Gly165Pro Thr58Gln + Ser203Asp + Thr212Ser Thr102Gln + Val202His + Thr214Ser Gly99Glu + Ile106Gly + Gly218Asn Ser97Asp + Leu125Val + Tyr213Ile Ile106Leu + Phe188Glu + Thr212Asn Thr58Pro + Gly153Pro + Asn162Gln Gly127Ser + Pro128Gln + Thr212Pro Tyr103Pro + Ser203Asp + Tyr205Ile Thr58Gln + Thr163Ser + Tyr205Ser Val94Cys + Ser215Glu + Gly218Pro Thr102Pro + Gly165Pro + Val202Thr Thr58Glu + Tyr103Asn + Pro200Gln Phe188Thr + Val202Asn + Asn217Ser Gly99Asn + Tyr205Ser + Ser215Glu Thr102Gly + Gly165Asp + Gly218Gln Gly60Asp + Ala186Pro + Tyr205Asn Thr102Asn + Ile106Asn + Ser158Glu Gly62Ser + Ser97Glu + Gly218Asn Ile106His + Ser129Glu + Thr212Asn Gly126Glu + Prt128Asn + Val202Asn Asp59Glu + Gly64Asn + Tyr213Ser Thr163Pro + Val204Thr + Tyr205Cys Ser129Glu + Asn162Ser + Leu216Cys Gly156Asn + Phe188Ala + Tyr205His Val94Ser + Gly101Ser + Leu125Asp Asn96Glu + Tyr166Ala + Leu216Cys Tyr103Gln + Phe188Met + Ser210Glu Gly101Ser + Thr212Pro + Thr219Pro Ser104Glu + Gly159Gln + Thr212Pro Gly62Asp + Gly126Ser + Leu216Pro Gly101Asp + Gly218Pro + Thr219Asn Ser157Glu + Tyr205Pro + Thr212Ser Pro209Glu + Ser210Glu + Gly218Ser Asn154Glu + Ser155Glu + Gly218Pro Asn211Glu + Thr212Asp + Leu216His Tyr205Gly + Ser215Glu + Leu216Asp Val202Ser + Ser215Asp + Leu216Asp Thr58Asp + Asp59Glu + Thr212Ser Tyr205Glu + Tyr213Pro + Ser215Asp Gly156Ser + Tyr205Asp + Ser215Glu Gly60Glu + Ser97Glu + Gly99Asn Ser155Glu + Asn162Ser + Ser190Glu Leu95Asn + Ser203Asp + Ser215Asp Ser203Asp + Ser215Asp + Thr219Gln Gly62Asn + Ser203Glu + Ser215Glu Ser203Glu + Tyr205Pro + Ser215Asp Phe188Asn + Ser203Asp + Ser215Asp Gly62Asn + Ser203Glu + Ser215Asp Ser203Glu + Ser215Asp + Leu216Ser Gly156Gln + Ser203Glu + Ser215Asp Ser203Asp + Tyr205Ala + Asn217Glu Tyr205Ala + Ser210Glu + Thr212Asp Ile164Cys + Ser210Glu + Thr212Asp Ser158Asp + Gln161Glu + Tyr205Pro Asp59Glu + Asn61Glu + Gly130Ser Gly64Gln + Ser98Glu + Ser100Asp Ser97Glu + Gly99Asp + Tyr213His Asn154Gln + Ser157Glu + Ser190Asp Ser158Asp + Ser160Glu + Tyr205Cys Gly201Pro + Ser203Glu + Tyr205Asp Gly99Gln + Ser129Asp + Tyr166Glu Ser160Asp + Asn162Glu + Phe188Gly Ser129Asp + Ser131Asp + Gly218Asn Asp59Glu + Gly165Asn + Pro209Glu Val94Gly + Ser215Asp + Asn217Glu Val202Glu + Tyr205Gln + Leu216Glu Gly156Glu + Ser158Glu + Tyr205Met Asn61Asp + Asn96Glu + Gly127Ser Gly159Asn + Phe188Asp + Gly218Asp Tyr103His + Asn162Glu + Ser190Asp Ser97Asp + Ser100Glu + Gly201Asn Ser157Glu + Ser160Glu + Tyr205Pro Gly153Asp + Val202Ser + Gly218Asp Ser157Glu + Gly159Glu + Ser190Asp Asp59Glu + Asn96Glu + Tyr205Ser Gly153Ser + Asn154Asp + Gly165Asp Asn154Glu + Gly165Asp + Asn211Ser Ser155Asp + Gly156Asp + Thr219Asp Ser210Asp + Tyr213Asp + Thr214Glu Asn61Glu + Ala186Gly + Pro209Asp Asn154Asp + Ser157Glu + Thr214Ser Ser100Glu + Leu125Glu + Tyr213His Gly60Glu + Leu95Glu + Ile106Leu Gly99Glu + Gly101Glu + Tyr103Glu Thr58Glu + Thr65Glu + Ile106Cys Ser97Glu + Gly101Asp + Val202Asn Gly62Asp + Ser210Asp + Leu216Ala Asn61Asp + Ser100Glu + Asn162Ser Gly127Glu + Asn162Glu + Gly165Glu Ile106Asn + Gly156Glu + Gly159Glu Ser100Glu + Gly126Glu + Ala186Gln Gly62Asp + Phe188Leu + Thr212Glu Ile106Gln + Ser215Glu + Gly218Glu Tyr103His + Ser155Glu + Gly218Glu Ala186Glu + Val202Glu + Ser215Asp Gly127Glu + Asn154Glu + Tyr213Thr Ser157Glu + Ser160Asp + Ser187Asp Gly62Asp + Leu216Glu + Thr219Gln Thr58Asp + Ser210Asp + Thr212Glu Ser155Glu + Ser157Asp + Gly218Asp Pro128Asp + Asn162Glu + Asn217Ser Gly156Asp + Gly165Glu + Tyr205Asn Ser158Glu + Phe188Asp + Val202Met Thr102Glu + Ser104Asp + Leu125Glu Ser98Asp + Ser100Asp + Ser104Glu Gly62Asp + Pro209Asn + Asn211Asp Ser160Glu + Ser187Asp + Ser190Asp Gly159Asp + Val202Asp + Ser203Asp Pro128Glu + Thr212Asp + Tyr213Asp Leu95Glu + Tyr205Asp + Ser215Asp Asp59Glu + Tyr205Glu + Ser215Glu Gly60Ser + Thr65Glu + Leu216Asp Gly101Ser + Asn154Glu + Ser158Glu Pro128Asp + Ser203Asp + Ser215Asp Ser203Asp + Ser210Glu + Ser215Glu Ser104Glu + Ser203Asp + Asn217Glu Ser160Asp + Ser203Asp + Leu216Asp Ser203Glu + Ser210Asp + Leu216Asp Gly127Glu + Ser129Glu + Ser215Glu Pro128Glu + Ala186Glu + Phe188Asp Asp59Glu + Asn61Glu + Ser203Asp Ser97Asp + Gly99Asp + Ser210Asp Asn96Glu + Ser100Asp + Thr212Glu Thr65Asp + Thr163Gln + Ser215Asp Ser98Asp + Thr102Glu + Leu216Gln Thr132Glu + Ser157Asp + Ser190Glu Ser129Glu + Ser157Asp + Ser190Glu Ser190Glu + Ser203Glu + Tyr205Glu Asn61Asp + Ser98Asp + Tyr205Asp Ser129Asp + Phe188Glu + Ser190Asp Ser100Glu + Ser104Glu + Tyr205Met Gly127Glu + Ser190Asp + Tyr205Leu Asp59Glu + Ser203Glu + Pro209Asp Gln161Glu + Ser215Glu + Asn217Asp Asp59Glu + Ser97Glu + Ser157Asp Leu125Glu + Gly153Glu + Ser203Glu Ser104Glu + Ser157Asp + Ser160Asp Asp59Glu + Asn96Glu + Ser187Glu Ser129Asp + Thr132Glu + Asn162Glu Ser131Glu + Ser203Glu + Gly218Glu Gly156Asp + Ser203Asp + Thr214Asp Ser160Glu + Thr163Asp + Ser215Asp Thr58Asn + Gly99Glu + Ala105Glu Asp59Glu + Phe188Leu + Thr212Asp Ser104Asp + Ser131Glu + Tyr205Glu Gly62Asp + Ser104Asp + Ser131Asp Ser104Glu + Ser131Glu + Gly156Glu Gly60Asp + Gly101Asp + Met198Gln Ser98Glu + Ser215Glu + Gly218Asp Tyr103Asp + Ser129Asp + Tyr205Glu Ser100Asp + Ile106Asp + Ser215Asp Thr65Asp + Ser100Glu + Ala105Glu Asp59Glu + Ser100Asp + Tyr205Ile Gly153Glu + Asn162Asp + Ser203Glu Ser98Glu + Thr212Glu + Ser215Glu Ser131Asp + Thr212Glu + Ser215Asp Ala105Asp + Thr212Glu + Ser215Asp Ser158Glu + Thr212Asp + Ser215Asp Ser157Asp + Ser203Glu + Thr219Asp Gly62Gln + Thr212Glu + Leu216Asp Ser97Glu + Ser187Glu + Asn217Asp Ser155Glu + Asn217Asp + Gly218Asn Gly159Glu + Ser190Glu + Asn211Glu Pro128Asp + Ser190Glu + Ser215Asp Asp59Glu + Gly156Asp + Ser160Glu Gly60Asp + Ser100Glu + Ser215Glu Gly130Glu + Gly153Glu + Tyr205Asn Thr102Glu + Gly153Glu + Asn217Asp Ser129Glu + Ser155Glu + Gln161Asn Tyr205Glu + Asn211Glu + Leu216Cys Gly153Glu + Gln161Asp + Ala199Asn Gly62Asn + Gly153Glu + Val202Asp Ser129Asp + Asn162Glu + Asn211Glu Gly153Glu + Ser158Asp + Leu216Asp Ser157Glu + Leu216Ile + Thr219Asp Ser158Glu + Ser215Asp + Thr219Asp

TABLE 34 Multi-loop Quadruple Mutation Variants Tyr103Cys + Val202Gln + Thr212Gly + Leu216Gln Asn61Gln + Gly101Asp + Ile106Ala + Tyr205Pro Asn61Ser + Gly64Gln + Thr132Gly + Gly165Gln Leu95Ile + Tyr205Glu + Tyr213Ala + Thr219Asn Gly126Ser + Val202Ser + Tyr205Cys + Asn217Glu Asn96Ser + Asn154Glu + Tyr166Thr + Tyr205His Gly101Ser + Ala105Gly + Phe188Gly + Gly218Asn Gly165Ser + Val202Gln + Tyr205Thr + Thr212Ser Gly126Asn + Ser190Glu + Pro209Asn + Leu216Val Ile106Glu + Gly127Ser + Asn154Ser + Tyr205Leu Thr58Pro + Ser203Asp + Tyr213Met + Leu216Val Gly62Gln + Asn96Glu + Ile164Asn + Leu216Ile Ser98Glu + Tyr166Gln + Thr212Asn + Leu216Val Gly156Asp + Tyr166Ala + Tyr208Pro + Leu216Ile Thr58Pro + Ser98Asp + Gly130Pro + Tyr205Val Gly130Asp + Phe188Asn + Leu216Gly + Gly218Pro Leu95Thr + Gly126Gln + Pro209Ser + Leu216Gly Val202Gln + Ser203Glu + Tyr205Asn + Gly218Asn Thr58Asn + Leu95Asn + Gly153Pro + Ser210Glu Gly127Ser + Ser131Glu + Thr132Asn + Gly165Asn Thr65Gly + Gly127Asn + Thr212Gln + Thr214Glu Asn96Gln + Gly126Asn + Gly127Ser + Val202Asn Gly64Ser + Ser160Asp + Asn211Ser + Thr212Gly Asp59Glu + Ala199Ser + Val202Ala + Tyr205Ser Leu125Gly + Thr132Gly + Pro200Gln + Tyr205Thr Gly159Ser + Thr163Glu + Ile164His + Tyr205Ser Gly127Pro + Phe188Met + Val202Gly + Ser215Glu Gly101Gln + Asn211Gln + Thr212Pro + Ser215Asp Pro128Gly + Pro200Ser + Thr214Gln + Leu216Gly Tyr205Cys + Thr212Pro + Leu216Met + Gly218Gln Thr102Pro + Val202Ser + Tyr205Asn + Gly218Asp Leu95Asp + Ile106Gln + Gly126Asn + Val202Ala Tyr103Gln + Ser203Asp + Thr212Asn + Leu216Asn Phe188Met + Ser210Glu + Thr212Pro + Thr219Pro Ser104Glu + Gly159Gln + Thr212Pro + Leu216Pro Gly60Gln + Ala186Gly + Thr212Gly + Ser215Asp Thr102Glu + Thr132Pro + Leu216Pro + Asn217Gln Leu95Ala + Phe188Thr + Tyr205Gln + Ser215Asp Thr58Gly + Gly60Asp + Val202Cys + Thr212Pro Gly156Asn + Val202Asp + Thr212Ser + Thr214Gly Thr58Pro + Gly165Glu + Tyr205Ser + Tyr213His Gly99Asn + Tyr166Asn + Ser203Asp + Leu216His Gly62Glu + Leu95Met + Ala105Ser + Gly126Ser Ile106Gln + Ser187Glu + Tyr205Gly + Thr219Pro Ser160Glu + Tyr205Ser + Thr212Pro + Leu216Thr Gly130Gln + Ser131Glu + Asn162Gln + Leu216Asn Gly156Glu + Thr163Ser + Asn211Ser + Thr212Asn Gly165Gln + Phe188Leu + Val202His + Thr212Glu Val94Cys + Pro128Asp + Val202Thr + Tyr205Ala Leu95Thr + Gly156Ser + Thr214Glu + Leu216Ala Gly99Pro + Tyr103Glu + Gly165Pro + Leu216Cys Gly64Asn + Thr65Glu + Tyr166Ala + Phe188Tyr Tyr103Gln + Gly156Pro + Tyr166Asn + Ser215Glu Gly126Gln + Pro128Gln + Val202Thr + Thr212Glu Gly64Ser + Val94Met + Gly130Asp + Tyr213Gly Gly99Asn + Val202Pro + Thr212Asn + Ser215Glu Gly101Pro + Asn154Ser + Leu216His + Asn217Glu Tyr103Asp + Gly153Pro + Ala186Asn + Val202Ser Leu125Thr + Gln161Asn + Thr212Gly + Leu216Asn Gly64Pro + Val94His + Ser210Asp + Thr212Gly Asn61Ser + Thr65Gln + Ser190Asp + Val202Gly Ala105Thr + Gly153Gln + Gly156Gln + Asn211Gln Asn61Gln + Tyr166Ile + Ala186Asn + Ser215Asp Gly60Gln + Tyr103Gly + Gly130Asn + Asn217Asp Leu95Pro + Tyr205Leu + Asn211Gln + Ser215Glu Thr102Asp + Gly126Gln + Val202Asn + Thr212Gly Val94Thr + Gly153Pro + Ala186Pro + Tyr205Glu Ser104Asp + Ala105Asn + Thr163Ser + Tyr205Ala Gly64Ser + Val94Asn + Gly130Asp + Leu216Cys Gly101Asn + Leu125Asn + Gly201Pro + Leu216Ser Gly159Pro + Gly165Pro + Ala186Thr + Thr212Pro Pro128Gln + Ser129Asp + Ala186Ser + Asn217Ser Gly60Asn + Gly159Ser + Phe188Val + Ser203Glu Ser98Glu + Gly99Asn + Thr102Asn + Ala186Pro Thr65Asn + Gly99Gln + Thr212Ser + Leu216Asp Val202Gln + Tyr205Thr + Tyr213Val + Ser215Glu Thr132Ser + Gly156Ser + Phe188Thr + Asn211Glu Leu125His + Gly156Ser + Thr212Glu + Leu216Asn Gly126Ser + Tyr205Asp + Thr214Gln + Leu216Asn Ala186Gly + Ser215Glu + Gly218Pro + Thr219Gln Thr58Gly + Gly60Asn + Leu125His + Leu216Gly Asp59Glu + Val94Gly + Tyr205Gln + Tyr213Ile Ser155Glu + Met198Thr + Val202Met + Tyr205Thr Gly127Asn + Tyr166Pro + Phe188Leu + Thr214Glu Leu95Cys + Gly130Gln + Thr212Ser + Leu216Val Asp59Glu + Thr65Gly + Leu95Gly + Leu216Asn Ser131Glu + Gly201Ser + Val202Gln + Tyr205Gly Leu125Val + Tyr205Gln + Ser215Asp + Gly218Gln Val94Gly + Thr102Ser + Ser203Glu + Leu216Asn Asp59Glu + Gly60Gln + Thr212Gly + Leu216Cys Ala186Gln + Gly201Gln + Val202Pro + Ser203Glu Thr65Ser + Ser98Asp + Gln161Ser + Tyr205Cys Tyr166Ala + Ser187Glu + Tyr205Gly + Leu216Val Gly99Asp + Met198Cys + Pro209Gly + Thr212Asn Thr65Pro + Ile106Gly + Tyr205Gln + Leu216Glu Leu95Ser + Thr102Asn + Leu216Asp + Asn217Ser Thr58Gly + Gly127Ser + Tyr166Glu + Val202Asn Val94Pro + Tyr213Thr + Thr214Glu + Asn217Gln Ser97Glu + Gln161Asn + Tyr166Gly + Val202Ser Tyr103Met + Thr132Gly + Tyr166Met + Ser210Asp Leu95Met + Gly156Asn + Val202Glu + Leu216Gln Ser97Asp + Met198Ala + Val204Thr + Tyr205Thr Gly101Ser + Gly153Pro + Asn217Glu + Thr219Pro Ile106Met + Thr163Glu + Tyr166Ile + Thr212Ser Gly165Ser + Ser190Asp + Pro200Ser + Val202Met Gly62Ser + Gly153Asp + Thr163Ser + Gly201Gln Gly101Gln + Thr102Ser + Gly153Ser + Val202Asp Val94Ala + Leu95Gly + Leu125Thr + Gly130Asp Asn96Asp + Tyr205Ala + Thr212Pro + Thr219Gly Gly153Gln + Tyr205Ile + Thr212Gly + Ser215Glu Ile106Cys + Asn154Glu + Tyr205Ala + Leu216Ala Asn96Asp + Gly99Pro + Tyr205Val + Asn217Gln Ala105Thr + Leu125Thr + Val204Ala + Tyr205Met Thr102Pro + Gly156Pro + Tyr208Val + Thr212Asn Gly60Ser + Phe188Ile + Thr212Ser + Leu216Asp Ser100Asp + Tyr103Gln + Gly159Gln + Leu216Val Pro128Gly + Gly165Gln + Ser203Glu + Leu216Ser Ser129Glu + Thr132Gln + Met198Thr + Thr219Gln Val94Pro + Gly99Asn + Thr132Gly + Gly159Asp Ser155Asp + Gly165Ser + Tyr205His + Thr212Gly Gly99Asp + Phe188Val + Thr207Asn + Leu216Pro Thr102Gln + Gly126Asn + Gly159Asn + Leu216Asp Asp59Glu + Thr132Pro + Thr163Pro + Phe188Thr Gly101Ser + Ala105Glu + Ala186Pro + Thr212Gly Ser129Asp + Gly153Asn + Gly159Pro + Val202Met Gly60Pro + Asn96Asp + Gly126Gln + Tyr166Ala Thr65Gln + Gly99Pro + Tyr205Ala + Ser215Asp Val94Gln + Gln161Asp + Tyr205Pro + Leu216Met Asp59Glu + Gly60Glu + Ala186Gly + Gly218Asn Ser98Asp + Gly99Glu + Phe188Met + Tyr213Ala Ser160Asp + Gln161Asp + Tyr205Ser + Thr219Gln Ala105Gly + Ile106Cys + Tyr205Asp + Ser215Asp Pro200Asn + Tyr205Asp + Asn211Gln + Ser215Glu Tyr205Asp + Thr207Ser + Thr214Ser + Ser215Asp Asn61Asp + Thr65Gln + Ser97Asp + Tyr205Ala Leu125His + Ser203Glu + Ser215Glu + Leu216Asp Thr65Asn + Ser203Glu + Tyr205Cys + Ser215Asp Gly64Pro + Ala186Ser + Ser203Asp + Ser215Asp Pro128Ser + Ser203Asp + Tyr213Ala + Asn217Glu Val202His + Tyr205Val + Ser210Glu + Thr212Asp Gly99Asn + Gly130Ser + Ser210Glu + Thr212Asp Tyr205Ala + Ser210Glu + Thr212Asp + Gly218Gln Phe188Ala + Ser203Asp + Tyr205Asp + Thr212Ser Asn154Glu + Ser155Asp + Thr163Asp + Tyr213Thr Gly99Gln + Ser129Asp + Tyr166Glu + Leu216Thr Thr102Gly + Ala105Thr + Ser155Glu + Ser157Asp Gly127Pro + Ser157Glu + Gly159Asp + Gln161Asp Gly101Asn + Asn154Asp + Thr163Gly + Gly218Glu Pro128Ser + Ser158Asp + Thr163Asp + Asn217Ser Ile106Val + Thr214Asn + Ser215Asp + Asn217Asp Thr58Gly + Asn154Asp + Ser157Asp + Ser190Glu Leu125Asn + Gly153Glu + Thr163Asp + Thr214Pro Gly64Pro + Ser157Glu + Gln161Glu + Tyr205Cys Ser155Asp + Ser187Asp + Ser190Glu + Thr212Gln Val94Pro + Ser155Asp + Ser157Asp + Ser187Glu Gly126Gln + Ser203Asp + Ser215Glu + Gly218Asp Gly156Ser + Ser187Asp + Ser190Glu + Thr212Ser Asn154Asp + Ser187Glu + Val202Asn + Leu216Ser Asn162Asp + Gly165Asp + Thr212Gln + Leu216Cys Ser155Glu + Ser187Asp + Pro200Ser + Tyr213Val Asn96Glu + Thr102Asp + Ala105Pro + Tyr213Val Asn154Ser + Gly156Glu + Asn162Asp + Thr212Ser Asn61Glu + Ser100Asp + Tyr205Val + Leu216Gly Thr65Gly + Asn154Glu + Ser157Asp + Asn162Glu Tyr205Leu + Thr212Glu + Ser215Glu + Leu216Glu Asn61Gln + Gly62Glu + Ser98Asp + Thr214Gln Gly62Glu + Ser98Glu + Gly130Ser + Val202Pro Thr65Glu + Tyr205Met + Thr212Asp + Thr214Gly Ala105His + Phe188Asp + Val202Asp + Asn211Ser Asp59Glu + Pro209Ser + Asn211Asp + Thr212Glu Leu95Gln + Gly126Gln + Gly127Asp + Ser155Asp Gly60Asp + Val94His + Asn154Ser + Ser210Glu Thr58Gly + Gly126Glu + Ser155Asp + Gly201Pro Gly156Asn + Tyr166Pro + Thr212Glu + Ser215Glu Thr212Asp + Ser215Glu + Leu216Ala + Thr219Ser Thr58Asp + Gly101Ser + Ser210Asp + Thr212Glu Ile106Glu + Gly126Asp + Gly127Asn + Leu216Pro Asn61Ser + Gly99Asp + Thr102Asp + Ser104Asp Ser210Asp + Thr212Asp + Tyr213Pro + Ser215Glu Gly127Pro + Pro128Ser + Ala186Glu + Ser203Asp Gly101Glu + Thr102Asp + Ser129Asp + Leu216Asn Gly62Glu + Leu95Asp + Gly159Asn + Leu216Gly Asn61Asp + Ser97Asp + Ser210Glu + Thr212Gln Ser203Glu + Tyr205Pro + Thr212Ser + Thr219Glu Thr65Glu + Tyr103Pro + Ser215Asp + Asn217Asp Gln161Asp + Thr163Glu + Ser187Glu + Asn211Gln Gly153Glu + Tyr166Gly + Ser187Glu + Leu216Met Asp59Glu + Gly159Glu + Ser160Asp + Pro209Gln Gly60Asn + Ser129Glu + Ser158Asp + Gly159Asp Gly130Glu + Ser131Asp + Ser155Glu + Thr212Asn Gly130Asp + Asn154Glu + Ser155Asp + Gln161Asn Ser129Asp + Gly130Glu + Gly156Glu + Gln161Ser Gly99Glu + Ser100Asp + Ser203Glu + Leu216Ser Pro128Asp + Ser129Asp + Val202Asn + Ser215Glu Tyr103Asp + Ser104Glu + Thr212Pro + Ser215Glu Thr65Pro + Ser157Glu + Ser158Asp + Ser215Asp Thr58Asn + Asn96Asp + Ser97Asp + Ser187Asp Ser104Asp + Ile164Gly + Ser215Asp + Leu216Asp Ser157Asp + Ser215Asp + Leu216Glu + Gly218Gln Gln161Ser + Ser187Asp + Ser215Glu + Leu216Glu Thr58Pro + Ser104Asp + Thr212Glu + Tyr213Glu Thr102Glu + Ser160Glu + Gln161Asp + Leu216Val Asn61Glu + Gly62Glu + Ser203Glu + Thr212Gly Tyr205Val + Ser210Asp + Asn211Glu + Leu216Glu Asp59Glu + Gly126Ser + Tyr205Glu + Ser215Glu Pro128Asn + Ser129Asp + Tyr205Asp + Ser215Asp Gly101Glu + Tyr205Glu + Ser215Asp + Leu216His Ser155Glu + Ile164Ser + Tyr205Asp + Ser215Asp Tyr103Ala + Tyr166Glu + Tyr205Glu + Ser215Glu Gly60Glu + Ser97Glu + Gly99Asn + Asn162Glu Ser187Glu + Ser203Glu + Tyr205Asp + Tyr213Pro Ser157Glu + Ser203Asp + Tyr208Pro + Ser215Glu Ser98Glu + Ser203Glu + Tyr205Val + Ser215Glu Gly127Gln + Ser160Glu + Ser203Asp + Ser215Glu Val94Glu + Ser203Asp + Ser215Asp + Leu216Pro Ser100Asp + Ser203Glu + Ser215Glu + Leu216Thr Ser157Glu + Phe188Thr + Ser203Asp + Ser215Asp Val94Gly + Leu95Glu + Ser203Glu + Ser215Asp Phe188Val + Ser190Asp + Ser203Asp + Ser215Glu Ser158Asp + Ser203Asp + Ser215Asp + Asn217Ser Ser157Asp + Ser203Asp + Ser215Asp + Thr219Gln Gly126Pro + Gly153Glu + Ser203Glu + Ser215Glu Ser131Glu + Gly165Asn + Ser203Glu + Ser215Asp Tyr103Glu + Gly153Asp + Ser190Glu + Tyr205Val Gly159Glu + Ser203Glu + Leu216Gln + Asn217Glu Ser100Asp + Gly153Gln + Thr163Asp + Ser190Asp Thr102Asp + Gly127Glu + Ser155Asp + Tyr213Pro Asn61Glu + Asn154Glu + Ser190Asp + Val204Met Asn154Asp + Gln161Ser + Ser190Asp + Ser203Glu Thr65Gly + Gly127Asp + Gly165Asp + Ser203Asp Asn96Asp + Gly99Glu + Gly101Asn + Ser187Glu Thr102Asp + Ala105Glu + Gln161Ser + Pro209Asp Gly127Glu + Ser129Glu + Gly130Ser + Ser160Asp Thr102Glu + Ser104Glu + Pro200Asn + Thr212Glu Ser98Asp + Ser100Glu + Pro128Gly + Gly159Asp Gly159Asp + Gln161Asp + Val204Gly + Ser215Glu Ser155Glu + Phe188Glu + Tyr205Asn + Ser215Glu Ser129Glu + Thr132Asn + Gly165Asp + Gly218Asp Ser100Glu + Gly153Asp + Gly165Gln + Thr219Asp Gly126Asp + Ser131Glu + Thr207Gly + Gly218Gln Asn61Asp + Gly99Glu + Tyr103Thr + Ser131Asp Gly99Asp + Ser203Asp + Tyr205Asp + Thr212Pro Ser190Glu + Ser203Glu + Tyr205Asp + Leu216His Gly60Pro + Ser157Asp + Ser203Glu + Tyr205Asp Ser157Glu + Thr163Glu + Pro209Ser + Ser215Glu Gly60Asn + Ser157Asp + Thr163Glu + Asn211Asp Thr102Glu + Gly127Asn + Gly130Glu + Tyr205Gln Asn61Glu + Gly153Ser + Pro209Asp + Ser215Glu Asp59Glu + Asn61Gln + Leu216Asp + Gly218Asp Leu125His + Ser129Asp + Ser131Asp + Val202Glu Ile106Cys + Tyr205Asp + Ser210Glu + Leu216Asp Ser97Glu + Gly156Asp + Thr163Asp + Thr212Gln Ser131Asp + Asn154Gln + Ser215Glu + Asn217Asp Ser104Glu + Thr212Ser + Ser215Asp + Asn217Glu Ser157Asp + Ser187Asp + Ser203Asp + Thr212Gly Asn61Asp + Asn96Asp + Ala105His + Asn162Asp Asn61Asp + Asn96Glu + Gly127Ser + Gly130Asp Thr58Gly + Ser158Glu + Asn211Asp + Tyr213Asp Ser158Asp + Asn162Asp + Thr212Asp + Leu216Thr Gly99Ser + Ser157Asp + Asn162Glu + Leu216Asp Gly159Gln + Asn162Glu + Ser190Asp + Ser215Glu Ser97Asp + Ser100Asp + Gly126Asn + Ser160Asp Gly60Glu + Ser98Glu + Gly153Gln + Ala186Asp Ser157Asp + Ser160Asp + Thr212Asn + Ser215Glu Gly62Pro + Ser131Glu + Gly165Glu + Thr219Asp Ser190Asp + Val202Glu + Ser215Glu + Leu216Ala Asn96Glu + Gly153Asp + Gly156Asp + Gln161Asn Tyr103Glu + Ser131Asp + Ser203Asp + Leu216Cys Gly130Glu + Ser157Asp + Ser187Asp + Tyr208His Asn61Asp + Thr163Gln + Ser203Asp + Thr214Glu Ser155Asp + Ser158Asp + Ser203Asp + Tyr205Thr Ser155Asp + Gly156Ser + Ser215Glu + Thr219Asp Gly60Gln + Thr65Gln + Asn96Asp + Leu125Glu Asp59Glu + Asn96Glu + Ser203Glu + Thr214Asn Asp59Glu + Asn96Asp + Ser215Asp + Leu216Ile Gly62Asp + Ser97Glu + Ala105His + Ser131Glu Leu125Asp + Gly165Asp + Ser203Asp + Tyr213Gln Leu95Glu + Tyr166Asn + Ser203Glu + Thr214Glu Gly101Asp + Asn162Asp + Gly165Glu + Leu216Asn Gly99Glu + Tyr103Glu + Ser129Glu + Tyr205Asn Asp59Glu + Asn96Ser + Thr212Glu + Ser215Asp Asn61Glu + Ser100Asp + Ala186Gly + Thr212Glu Thr65Gly + Ser97Glu + Gly101Glu + Gly130Glu Ala186Glu + Phe188Tyr + Thr212Asp + Asn217Asp Asn61Gln + Gly156Glu + Ser215Glu + Thr219Glu Gly99Gln + Ser129Glu + Ser155Glu + Gly218Glu Ser160Glu + Thr163Asp + Thr212Ser + Ser215Asp Asn96Asp + Thr102Asp + Ser157Glu + Ala186His Gly99Glu + Ala105Asp + Thr214Asn + Leu216Val Asn162Gln + Ala186Asp + Tyr205Ser + Leu216Asp Ser97Glu + Gly156Glu + Gly159Asp + Val202Asn Ile106Cys + Ser129Glu + Ser155Glu + Ala186Glu Ser100Glu + Leu125Gly + Gly127Glu + Ser203Asp Thr65Asp + Ser131Asp + Ile164Asn + Thr212Asp Ser97Glu + Thr102Glu + Gln161Ser + Tyr205Leu Asp59Glu + Gly99Glu + Ser203Asp + Tyr205Asn Asp59Glu + Gly99Asp + Gly126Ser + Thr214Glu Ser160Asp + Pro209Asn + Ser215Asp + Gly218Glu Ser155Glu + Gly159Asn + Pro209Glu + Gly218Glu Ser155Asp + Asn211Asp + Tyr213Met + Gly218Asp Ser129Asp + Thr132Glu + Ser160Glu + Ala186Ser Val94Glu + Ser97Asp + Gly101Pro + Ser203Glu Gly101Asp + Gly126Asp + Thr132Asp + Thr212Asn Ser98Glu + Gly127Glu + Thr163Asp + Thr212Asn Gly99Asp + Gly101Pro + Thr102Glu + Ser203Glu Gly159Asp + Thr212Asp + Ser215Asp + Asn217Ser Ser158Glu + Asn162Gln + Thr212Glu + Ser215Asp Val94Pro + Ser155Asp + Thr212Glu + Ser215Glu Gly99Gln + Ile106Asp + Thr212Glu + Ser215Glu Thr58Asp + Tyr205Pro + Thr212Glu + Ser215Asp Asp59Glu + Ser98Glu + Ser203Asp + Tyr205Gly Asp59Glu + Ser100Asp + Ser104Asp + Ala105Pro Asp59Glu + Ser100Asp + Ser104Asp + Tyr205Ile Pro128Glu + Asn162Asp + Thr212Glu + Leu216Cys Asn96Gln + Thr102Asp + Ser129Glu + Asn162Glu Ser97Glu + Gly130Asn + Ser155Asp + Ala186Glu Ala105Glu + Ser155Glu + Gln161Asp + Tyr205Thr Gly62Asp + Ser158Asp + Ser215Asp + Leu216Asn Thr102Asp + Gly126Asp + Ser158Asp + Thr212Ser Thr58Asn + Gly64Pro + Thr65Glu + Val94Asp Ser190Glu + Ser203Glu + Tyr213Glu + Leu216His Gly159Asp + Ser203Asp + Tyr205Cys + Tyr213Asp Ile106Asp + Gly130Asp + Ser215Asp + Asn217Gln Ser98Glu + Thr102Asp + Ser203Asp + Thr214Gly Gly99Gln + Ser104Asp + Gly130Asp + Asn154Glu Ser104Glu + Gly126Ser + Gly130Asp + Asn217Glu Asn96Gln + Ser100Glu + Ser104Glu + Tyr205Asp Asn61Gln + Gly101Asp + Ser157Asp + Gly165Glu Thr102Ser + Val202Cys + Thr214Glu + Gly218Glu Asn61Gln + Ser104Glu + Gly127Glu + Gly165Pro Thr65Gly + Tyr205Glu + Asn211Glu + Leu216Cys Gly99Gln + Gly159Ser + Pro209Asp + Leu216Asp Asp59Glu + Ser158Asp + Tyr205Pro + Thr212Glu Asp59Glu + Ser131Glu + Thr212Glu + Leu216Ile Ala105Asp + Leu125Glu + Asn211Glu + Leu216Ile Thr58Asp + Gly156Ser + Asn211Asp + Ser215Glu Ser157Glu + Gly159Ser + Gly165Pro + Thr219Glu Asn162Asp + Tyr205Gln + Thr212Glu + Leu216Asp Ser100Glu + Met198Gln + Thr212Asp + Leu216Glu Gly64Gln + Pro128Glu + Thr132Asp + Thr163Gly Gly62Gln + Pro128Asp + Thr132Asp + Leu216Thr Gly101Asp + Ile106Met + Leu125Gly + Pro128Glu Thr65Glu + Asn96Glu + Gly156Asp + Leu216Cys Ser104Asp + Leu125Gln + Ser129Asp + Asn162Gln Asp59Glu + Gly130Glu + Gly153Glu + Tyr205Asn Ser129Asp + Ser155Asp + Tyr205Val + Leu216Asp Ser129Glu + Ser155Glu + Gln161Asn + Ser210Glu Gly60Asp + Gly127Asp + Asn162Glu + Phe188Asn Gly62Gln + Ser98Glu + Ser187Asp + Ser203Glu Ser104Asp + Gly130Gln + Ser187Asp + Ser203Glu Asn61Asp + Tyr103Ser + Thr212Asp + Thr219Asp Asp59Glu + Gly101Asp + Gly159Pro + Gln161Glu Gly60Glu + Leu125Ser + Gly153Glu + Val202Asp Thr132Glu + Ser157Glu + Thr212Gly + Thr219Asp Gly101Glu + Leu125Cys + Gly130Glu + Thr219Asp Gly60Asp + Pro128Glu + Ser157Asp + Tyr205Cys Pro128Glu + Asn154Gln + Ser157Asp + Ser203Asp Ser97Asp + Ala105Asp + Ser203Asp + Thr212Gln Ser100Asp + Ser158Glu + Tyr166Asp + Phe188Pro Tyr205Thr + Asn211Glu + Ser215Glu + Leu216Asn

TABLE 35 Multi-loop Quintuple Mutation Variants Tyr103Cys + Gly126Gln + Gly156Glu + Val202Gln + Thr212Gly Val94Met + Gly101Asn + Thr102Asn + Tyr205Leu + Gly218Asp Ser98Asp + Tyr103His + Leu125Cys + Gly159Asn + Met198Cys Leu95Met + Gly101Pro + Tyr103Ala + Gly165Ser + Leu216Asp Thr65Asp + Val94Thr + Thr132Gly + Asn154Ser + Thr219Gly Val94Thr + Thr132Gly + Ala186Gln + Tyr205Cys + Thr214Gln Thr163Gln + Tyr205Cys + Thr212Pro + Leu216Met + Gly218Gln Leu95Asp + Gly101Gln + Ile106Gln + Gly126Asn + Val202Ala Gly99Glu + Gly126Ser + Tyr166Ala + Asn211Gln + Leu216Asn Gly60Asn + Gly159Glu + Gln161Ser + Asn162Gln + Leu216Ser Ala186His + Val202His + Thr212Pro + Ser215Glu + Leu216Thr Gly99Gln + Leu125Ile + Gly127Ser + Tyr205Val + Thr212Asp Thr58Gly + Val94Gly + Tyr166Cys + Ser203Asp + Tyr205His Val94Asp + Gly99Asn + Gln161Ser + Gly201Ser + Thr212Asn Gly126Asn + Gly127Pro + Gly153Asp + Gly159Pro + Ile164Ala Asn61Glu + Thr102Asn + Gly165Gln + Phe188Leu + Val202His Val94Cys + Gly156Ser + Val202Thr + Tyr205Ala + Leu216Ala Tyr103Gln + Gly126Gln + Gly156Pro + Tyr166Asn + Ser215Glu Gly99Asn + Val202Pro + Thr212Asn + Tyr213Gly + Ser215Glu Gly101Asp + Leu125Thr + Gln161Asn + Thr212Gly + Leu216Asn Asn61Ser + Gly153Gln + Gly156Gln + Val202Gly + Asn211Gln Asn61Gln + Ala105Thr + Tyr166Ile + Ala186Asn + Ser215Asp Ala105Gln + Leu125Gly + Thr163Ser + Val202Gln + Asn217Asp Pro200Ser + Val202Ser + Ser203Glu + Thr207Gly + Thr214Gly Val94Asn + Gly130Pro + Tyr205Ser + Ser215Asp + Thr219Pro Thr163Asn + Ala199Gln + Tyr205Asn + Thr212Ser + Ser215Glu Leu95Met + Gly156Asn + Val202Cys + Leu216Gln + Gly218Glu Val94His + Gly99Glu + Asn211Ser + Tyr213His + Leu216Met Gly64Ser + Val94Gln + Ser157Asp + Tyr205Ser + Thr207Pro Ile106Ala + Gly165Asn + Ser190Glu + Ala199Asn + Tyr205Asn Asp59Glu + Gly159Ser + Gly165Asn + Phe188Thr + Asn217Gln Gly130Pro + Ser155Glu + Gly159Asn + Tyr205Pro + Thr212Ser Tyr103Gln + Ser155Glu + Val202Asn + Thr212Ser + Thr219Asn Gly60Pro + Val94Ser + Ser100Glu + Tyr103Leu + Tyr205Ser Gly60Gln + Leu95Val + Pro128Gly + Ser158Asp + Tyr205Thr Thr132Gln + Asn162Ser + Gly165Asp + Tyr205Gly + Tyr208Cys Gly62Asn + Val94Thr + Ala105His + Pro128Glu + Tyr205His Thr58Pro + Gly156Pro + Val202Pro + Thr212Glu + Leu216Thr Leu95Met + Gly99Gln + Tyr103Cys + Asn154Gln + Thr212Gln Ser98Glu + Tyr205Asn + Pro209Gln + Tyr213His + Thr219Ser Gly62Ser + Val94His + Tyr103Gln + Ser187Glu + Phe188Tyr Gly62Gln + Asn96Ser + Gly165Glu + Val202Gly + Tyr205Thr Gly60Pro + Ile106Thr + Ser160Glu + Thr214Gln + Leu216Ile Ser100Asp + Ala105Thr + Tyr205Ile + Thr214Gly + Leu216Cys Leu95Pro + Ala105Asn + Phe188Leu + Thr212Pro + Leu216Val Asp59Glu + Gly60Ser + Gly127Pro + Tyr205Thr + Leu216Ala Ala105Asn + Ser131Asp + Thr163Pro + Leu216His + Gly218Asn Leu95Cys + Tyr103Met + Ile106Cys + Ala186Asp + Tyr205Met Leu125Cys + Tyr205His + Leu216Asn + Asn217Ser + Thr219Asn Asn96Ser + Gly153Glu + Asn154Gln + Ala186Gln + Thr212Asn Gly101Gln + Ala105Pro + Gly130Asp + Asn154Gln + Gly156Asn Thr58Gly + Gly159Pro + Thr163Ser + Tyr205Gln + Thr212Asn Tyr205Pro + Thr212Asn + Tyr213Gln + Ser215Asp + Leu216Gln Gly130Pro + Gly153Pro + Gln161Glu + Tyr205Met + Thr212Gln Ala105Gly + Ser157Asp + Tyr166Asn + Val202Gln + Thr212Ser Thr58Gln + Asn96Ser + Ser203Asp + Thr207Gly + Leu216Ser Ser158Glu + Val202Ser + Thr212Asn + Thr214Gln + Thr219Asn Asn61Ser + Ser100Glu + Tyr205Leu + Thr212Ser + Asn217Ser Gly60Gln + Gly99Glu + Thr212Ser + Tyr213Gln + Leu216Thr Leu125Asn + Ser129Asp + Asn162Ser + Ala186His + Tyr205Gln Gly64Gln + Leu95Glu + Ala199Asn + Val204His + Leu216Met Leu95His + Gly101Pro + Val204His + Tyr205Asn + Leu216Gln Thr163Ser + Gly165Asn + Pro209Gly + Tyr213His + Leu216Asp Gly127Gln + Ser131Asp + Thr132Pro + Tyr205Cys + Leu216Asn Asn61Gln + Gly101Pro + Ser190Asp + Tyr205Pro + Thr214Gly Gly126Asn + Pro200Gly + Val204Gly + Tyr205Asp + Thr212Asn Asn61Gln + Val94Gln + Gly126Asp + Tyr205Val + Thr212Gln Gly60Gln + Ile106Pro + Ser131Glu + Ile164Ala + Val202Pro Gly101Ser + Tyr103Glu + Ser104Asp + Gly165Asn + Pro209Gln Thr58Glu + Asp59Glu + Tyr166His + Val202Cys + Thr219Asn Gly126Pro + Ala186His + Tyr205Asp + Tyr213Val + Ser215Asp Gly60Asp + Ser97Glu + Ala105Pro + Leu125Cys + Gly153Pro Asn154Ser + Pro209Ser + Thr214Glu + Ser215Asp + Leu216Glu Thr163Pro + Ser203Glu + Val204Thr + Tyr205Cys + Ser215Asp Gly62Ser + Met198Gln + Ser203Glu + Ser215Asp + Leu216His Gly156Asn + Ser203Asp + Tyr205Met + Ser215Asp + Gly218Pro Gly60Gln + Pro128Ser + Ser203Glu + Ser215Glu + Leu216Pro Tyr166Cys + Tyr205Glu + Pro209Asn + Ser215Glu + Leu216Asp Tyr103Leu + Leu125Gly + Gly153Glu + Ser190Glu + Leu216Ile Gly99Pro + Ile106Thr + Gly153Asp + Ser155Asp + Ser190Asp Thr65Pro + Leu125His + Ser210Glu + Thr212Asp + Tyr213Ala Gly64Ser + Thr65Ser + Gly99Asn + Pro209Glu + Asn211Glu Leu95Asn + Gly101Asn + Asn154Asp + Gly156Glu + Tyr166Gln Ser155Glu + Thr163Asp + Tyr205Cys + Thr212Pro + Leu216Val Gly159Pro + Thr163Ser + Ser203Asp + Tyr205Asp + Thr212Ser Thr102Gln + Gly126Asp + Gly153Asp + Phe188Thr + Tyr205His Asn61Ser + Val202Glu + Ser203Glu + Tyr205Asp + Thr212Pro Thr58Gln + Thr102Pro + Tyr213Cys + Ser215Glu + Asn217Asp Gly60Asp + Gly62Glu + Thr65Glu + Gly126Asn + Leu216Thr Ala105Gly + Gly126Pro + Gly153Glu + Leu216Thr + Gly218Asp Tyr103Gln + Ala105Thr + Val202Asp + Tyr205His + Ser215Glu Val202Glu + Tyr205Thr + Pro209Asn + Ser215Asp + Leu216Met Val94Asn + Ala105Asn + Ile106Val + Ser203Glu + Gly218Asp Asn96Ser + Ser203Asp + Tyr205Ile + Thr212Gln + Gly218Asp Asn61Asp + Gly62Gln + Thr65Asp + Gly126Asn + Gly159Pro Gly153Gln + Ala186Glu + Tyr205Thr + Leu216Thr + Asn217Asp Leu125Asp + Gly126Asp + Ala186Ser + Gly218Glu + Thr219Pro Thr58Glu + Thr65Glu + Val94Gln + Gly101Pro + Gly165Gln Gly153Glu + Ser158Glu + Thr163Glu + Gly165Pro + Leu216Met Gly159Asp + Thr163Glu + Tyr166Thr + Ala186His + Pro200Ser Leu125Thr + Gly130Ser + Asn154Asp + Ser157Asp + Ala186Glu Asp59Glu + Asn96Glu + Gly101Glu + Pro128Ser + Thr212Asn Asp59Glu + Asn96Asp + Gly101Asp + Tyr166Asn + Thr219Asn Gly99Glu + Gly101Glu + Gly127Glu + Tyr166Leu + Asn217Gln Ala186Gln + Ser187Asp + Leu216Pro + Gly218Glu + Thr219Gln Thr58Pro + Leu95Ile + Gly126Glu + Gly153Glu + Phe188Asp Thr58Glu + Asn96Glu + Gly99Asp + Phe188Met + Gly218Gln Gly62Glu + Asn162Ser + Val202Met + Thr212Asp + Leu216Met Gly62Pro + Gly99Gln + Ser104Glu + Ser131Asp + Tyr213Thr Gly62Glu + Gly130Ser + Tyr205Glu + Ser215Glu + Leu216Asn Gly126Gln + Gly127Glu + Asn154Glu + Tyr213Pro + Leu216His Ser129Glu + Asn154Glu + Asn162Ser + Gly165Asp + Leu216Cys Thr58Gly + Leu125Asn + Gly156Asp + Gln161Glu + Tyr205Gly Gly126Ser + Gly153Gln + Gly156Glu + Gln161Asp + Tyr205Pro Thr163Gln + Val202His + Tyr205Asp + Pro209Gly + Thr212Asp Gly62Gln + Val202Met + Asn211Asp + Thr212Asn + Thr214Glu Thr102Gln + Gly126Pro + Thr212Asp + Ser215Glu + Leu216Asn Gly101Ser + Gln161Asn + Thr212Glu + Ser215Asp + Leu216Gln Leu125Gln + Ala186Glu + Val202Cys + Ser203Asp + Ser215Aso Ser129Glu + Ser155Glu + Thr163Asp + Ala186Ser + Tyr208Ser Thr58Asn + Leu125Glu + Pro128Glu + Thr163Ser + Gly165Pro Asn61Glu + Gly62Asp + Gly130Ser + Asn211Asp + Thr212Gly Thr65Gln + Asn154Asp + Ser215Glu + Leu216His + Asn217Glu Asp59Glu + Ser97Glu + Thr102Gln + Ser210Asp + Leu216Thr Gly153Glu + Ile164Thr + Val202Gln + Ser203Glu + Asn217Glu Val94His + Leu125Cys + Gly126Ser + Ser155Glu + Gln161Glu Ser187Glu + Tyr205Thr + Thr212Asn + Tyr213Gly + Asn217Glu Gly99Ser + Gly130Glu + Asn162Asp + Thr163Asp + Tyr205Asn Pro128Gln + Gly156Asp + Ala186Glu + Ser203Glu + Tyr213Gln Gly62Asp + Gly156Asn + Ala186Pro + Thr212Glu + Leu216Asp Gly126Ser + Tyr166Gly + Ser203Asp + Pro209Asp + Ser215Asp Tyr103Val + Pro128Asp + Gly153Asn + Asn162Ser + Ser190Asp Gly130Pro + Gly156Asn + Gln161Asp + Thr163Glu + Ser187Glu Leu125Pro + Ser203Asp + Pro209Gln + Thr212Glu + Leu216Asp Leu95Asp + Gly126Glu + Pro128Gly + Gly159Gln + Ala186His Gly62Ser + Ser190Asp + Thr214Gly + Ser215Asp + Gly218Asp Gly64Asn + Tyr103Ile + Leu125Asp + Ser203Asp + Asn217Glu Gly126Ser + Gln161Asp + Asn162Ser + Thr214Glu + Ser215Glu Ile106Leu + Gly126Asp + Gly127Glu + Gly130Gln + Val202Glu Asp59Glu + Thr102Ser + Ala105Gly + Gly159Glu + Ser160Glu Leu95Asp + Asn96Asp + Tyr166Leu + Val204Ala + Gly218Glu Gly130Glu + Ser131Asp + Gly153Gln + Ser158Glu + Ala199Pro Gly60Glu + Asn61Asp + Gln161Asn + Pro209Gln + Ser215Glu Thr65Asn + Gly99Gln + Asn154Asp + Pro209Asp + Ser210Glu Asn154Glu + Ser155Asp + Tyr213Thr + Ser215Glu + Gly218Pro Ser100Asp + Asn154Glu + Ser155Asp + Pro209Ser + Thr214Gly Gly62Asn + Gly153Asp + Asn211Glu + Thr212Asp + Gly218Gln Tyr103His + Ser129Glu + Tyr205Pro + Asn211Asp + Thr212Glu Ser129Asp + Asn162Gln + Tyr205Leu + Asn211Asp + Thr212Asp Ser100Glu + Gly153Pro + Thr163Pro + Val202Glu + Ser203Glu Thr58Asp + Thr65Pro + Val202Glu + Ser203Asp + Leu216Asn Thr65Pro + Gly126Asn + Ser157Glu + Ser158Asp + Ser215Asp Gly101Asn + Ser157Asp + Ser158Glu + Ser215Glu + Leu216Ile Asn96Glu + Ser97Glu + Gly159Asn + Phe188His + Ser210Glu Thr65Asn + Ser98Asp + Gly159Asn + Ser215Asp + Leu216Asp Asn96Glu + Pro128Ser + Tyr205Cys + Ser215Glu + Leu216Glu Ile106Gly + Ser155Glu + Thr212Asn + Ser215Glu + Leu216Asp Leu125Glu + Thr163Asn + Val202Thr + Ser215Asp + Leu216Glu Gly101Gln + Gly130Glu + Tyr205Ala + Ser215Glu + Leu216Glu Pro128Gly + Ser210Asp + Thr212Ser + Ser215Glu + Leu216Glu Ser104Asp + Ala105Glu + Met198Pro + Ser203Glu + Leu216Ser Ser97Glu + Ser98Glu + Ala105Gly + Gly153Asp + Asn217Gln Gly153Gln + Ser210Asp + Asn211Asp + Thr212Ser + Ser215Asp Thr65Glu + Leu125Cys + Gly126Ser + Val202Glu + Ser215Asp Thr102Gln + Gly165Asn + Ser203Glu + Tyr205Glu + Pro209Asp Leu95Glu + Asn96Gln + Tyr205Glu + Pro209Ser + Ser215Glu Gly126Asn + Tyr205Asp + Ser210Asp + Ser215Glu + Leu216Met Gly62Gln + Leu95Pro + Ser158Asp + Tyr205Asp + Ser215Glu Asn61Glu + Gly101Glu + Gly126Ser + Leu216Met + Thr219Ser Gly156Asp + Ala186Gln + Ser190Glu + Ser203Glu + Thr212Pro Gly60Glu + Ser97Glu + Gly99Asn + Asn162Glu + Asn217Ser Thr58Pro + Ser160Asp + Ser190Asp + Val202Asn + Thr212Asn Gln161Asn + Ser203Asp + Asn211Glu + Tyr213Cys + Ser215Glu Pro128Glu + Gly165Gln + Ala186His + Ser203Glu + Ser215Glu Gly101Pro + Thr132Ser + Ser158Glu + Ser203Glu + Ser215Glu Pro128Ser + Ala186Gly + Ser203Glu + Ser210Glu + Ser215Asp Asn61Gln + Ser158Asp + Ala186Ser + Ser203Asp + Ser215Glu Leu95Val + Ser100Asp + Tyr166Met + Ser203Glu + Ser215Asp Gly153Glu + Ala186Gln + Ser203Glu + Ser215Asp + Thr219Gly Ser160Asp + Ser203Glu + Val204Gln + Thr212Pro + Ser215Glu Thr58Pro + Leu95His + Ser155Asp + Ser203Asp + Ser215Asp Asn154Gln + Ser157Asp + Ser203Glu + Thr212Ser + Ser215Glu Leu95Gln + Ser100Asp + Ser203Glu + Tyr205His + Ser215Glu Asn154Asp + Tyr166His + Ser203Asp + Tyr205Ala + Ser215Glu Ser98Asp + Ser203Glu + Asn211Ser + Ser215Asp + Leu216Ser Asn162Glu + Ser203Asp + Tyr208Asn + Thr212Ser + Ser215Asp Gln161Asp + Thr163Gln + Ser203Glu + Ser215Glu + Leu216Thr Leu95Cys + Ser129Glu + Ser203Asp + Asn211Gln + Ser215Asp Thr132Asn + Asn154Glu + Ser203Asp + Thr212Asn + Ser215Asp Pro128Gln + Ser131Asp + Gly156Asn + Ser203Glu + Ser215Asp Thr58Glu + Asn61Ser + Gly99Pro + Ser203Glu + Ser215Asp Ser100Asp + Gly153Ser + Ser203Asp + Tyr205Val + Ser215Glu Ala105Glu + Ser203Asp + Thr212Asn + Ser215Glu + Gly218Asn Thr102Asn + Ser104Asp + Gly127Asn + Ser203Asp + Ser215Asp Gly62Asp + Leu125Thr + Gly153Glu + Gly165Ser + Ser190Glu Gly153Glu + Phe188His + Ser190Glu + Ser203Glu + Leu216Gly Thr58Asp + Phe188Asn + Ser203Glu + Tyr205Leu + Asn217Asp Thr65Gly + Thr102Ser + Gly126Asp + Thr212Ser + Gly218Asp Asp59Glu + Thr163Glu + Ser190Glu + Thr212Gln + Leu216Ser Tyr166Val + Ser203Glu + Ser210Asp + Thr212Ser + Leu216Asp Gly159Asp + Ser203Glu + Thr207Pro + Thr212Asn + Leu216Asp Ser157Asp + Asn162Gln + Gly165Gln + Ser203Glu + Leu216Asp Leu95Thr + Ser131Glu + Val202Ser + Ser203Asp + Leu216Glu Ser100Asp + Gly127Asn + Ser203Asp + Tyr208Val + Leu216Asp Thr102Gly + Ser129Glu + Ser203Asp + Tyr205Ala + Leu216Asp Thr65Gln + Ser98Asp + Thr102Pro + Gly126Asp + Gly165Asp Thr65Asn + Gly127Glu + Ser129Asp + Tyr205Ile + Ser215Glu Gly60Asn + Ser203Glu + Tyr205Val + Ser210Asp + Thr212Glu Val94Cys + Ser98Glu + Pro209Ser + Ser210Asp + Thr212Glu Gly60Gln + Tyr103Asn + Pro128Glu + Ser157Asp + Gly159Asp Ser98Glu + Ser100Asp + Ser203Glu + Thr212Gln + Leu216Thr Gly60Glu + Thr65Asp + Thr132Gln + Asn154Ser + Leu216Asp Asn96Asp + Ser100Asp + Thr102Pro + Tyr205Asp + Thr212Gln Thr102Gly + Phe188His + Gly201Asn + Tyr205Glu + Gly218Asp Asn154Gln + Ser155Asp + Tyr205Gln + Pro209Glu + Thr212Asp Tyr103Val + Gly153Asn + Pro209Asp + Thr212Asp + Gly218Glu Thr65Glu + Ile106Met + Leu125His + Tyr205Leu + Ser215Asp Leu125Met + Gly156Asp + Tyr166Ser + Ser187Glu + Ser215Asp Thr58Glu + Gly60Glu + Ser131Glu + Tyr205Val + Gly218Pro Thr58Asp + Gly60Asp + Asn61Ser + Asn162Gln + Ser203Asp Ser157Glu + Ser203Asp + Tyr205Asp + Thr212Ser + Leu216Cys Thr58Asn + Thr102Gln + Ser155Glu + Ser157Asp + Thr212Glu Ala105Gln + Ser155Asp + Ser157Asp + Ser203Asp + Thr212Gly Thr163Glu + Val202Asp + Tyr205Cys + Leu216Pro + Gly218Glu Thr58Gln + Asn61Glu + Ile106Thr + Pro209Asp + Ser215Asp Gly62Glu + Ser97Glu + Asn154Ser + Tyr205Asp + Tyr213Val Leu125Gln + Gly130Asp + Leu216Glu + Gly218Asp + Thr219Gly Gly126Ser + Ser131Glu + Tyr205Ala + Leu216Asp + Gly218Glu Ile106Ala + Ser160Glu + Asn162Glu + Ala186Gly + Ser203Asp Asp59Glu + Thr65Asp + Asn162Asp + Leu216Ile + Asn217Ser Asn96Gln + Thr102Pro + Gly127Glu + Ala186Pro + Ser190Asp Gly99Asn + Leu125Thr + Ser157Glu + Gly165Glu + Thr212Gln Val94His + Gly126Asn + Ser157Glu + Gly165Glu + Tyr205Val Asp59Glu + Asn61Ser + Val202Asn + Thr207Pro + Asn211Asp Asp59Glu + Tyr103Ala + Ser155Glu + Pro209Glu + Thr212Asn Gly101Gln + Gly127Glu + Gly153Ser + Asn154Glu + Ser158Asp Ser97Glu + Thr212Ser + Ser215Asp + Leu216Cys + Asn217Glu Gly99Gln + Ser157Glu + Ser215Asp + Leu216Ser + Asn217Asp Ser131Glu + Gly159Asn + Tyr205Met + Ser215Glu + Asn217Glu Val94Ala + Gly101Asp + Gly156Gln + Ser215Asp + Asn217Asp Asn61Asp + Asn96Glu + Pro209Ser + Ser215Glu + Leu216Ala Asn61Glu + Asn96Glu + Gly127Ser + Ser203Glu + Thr214Ser Gly99Pro + Ser100Asp + Thr102Glu + Ala105Gly + Asn211Glu Ser155Glu + Ala186Pro + Tyr205Asp + Leu216Cys + Gly218Glu Phe188Asp + Ser203Asp + Tyr213Ser + Thr214Glu + Gly218Asn Ser129Asp + Thr163Gly + Gly165Glu + Ser203Asp + Tyr205Ile Ser104Asp + Ser158Glu + Asn162Glu + Val202Gln + Leu216Val Ser98Asp + Ser158Asp + Asn162Glu + Leu216Ile + Thr219Gly Pro128Gly + Ser158Asp + Asn162Glu + Val202Gln + Asn217Glu Thr102Glu + Ala105Asn + Ser129Asp + Tyr208Thr + Leu216Gln Thr65Asn + Ser157Asp + Asn162Glu + Ser203Asp + Tyr205Gly Ala105Gly + Ser157Asp + Ser160Glu + Ala186His + Ser210Asp Ser98Glu + Gly153Asp + Val202Ser + Tyr205Asn + Gly218Asp Gly62Asp + Gly64Asn + Gly99Pro + Gly127Asp + Gly153Glu Thr58Ser + Val94Met + Ser104Glu + Gly153Asp + Tyr166Asp Asn61Asp + Thr65Gly + Ala186Gln + Ser210Asp + Thr214Asp Gly127Asn + Val202Asp + Ser210Glu + Ser215Glu + Asn217Ser Gly62Asn + Gly101Asp + Tyr103Met + Ser104Glu + Ser203Glu Gly159Asp + Asn162Glu + Ile164Ala + Thr212Asn + Ser215Asp Ser98Asp + Gly101Glu + Asn154Gln + Tyr205Gln + Ser210Asp Leu95Glu + Tyr166Asn + Phe188Met + Ser203Glu + Thr214Glu Gly101Pro + Ser129Glu + Ser203Asp + Thr214Glu + Gly218Gln Asn96Gln + Asn162Glu + Gly165Glu + Val202Asp + Tyr205Met Thr65Gln + Val94Asp + Ser104Glu + Leu125Asn + Thr212Asp Thr102Pro + Ser155Asp + Ser187Glu + Tyr205Val + Ser215Glu Asn96Glu + Thr102Gln + Ala186Asp + Phe188Gln + Ser190Asp Thr58Glu + Asn61Glu + Gly64Asn + Ala105Pro + Asn217Glu Val94Ala + Gly126Asp + Asn154Glu + Tyr166Pro + Thr212Glu Thr58Asn + Asn61Ser + Gly130Glu + Phe188Glu + Asn217Asp Thr58Asp + Ser97Asp + Ile106Leu + Gly153Gln + Gly156Asp Thr58Asp + Ser97Glu + Thr102Pro + Ala105Ser + Ser158Asp Tyr103Glu + Gly127Glu + Asn154Gln + Ser157Asp + Thr163Ser Asn96Gln + Pro128Glu + Gly130Gln + Ser131Glu + Ser157Asp Gly127Glu + Gly153Asn + Pro209Asp + Thr214Glu + Thr219Ser Ser100Asp + Ile106Met + Pro128Asn + Ser190Asp + Gly218Glu Thr58Asp + Asn96Asp + Gly127Asp + Tyr166Ala + Ala186Ser Asn96Glu + Thr163Pro + Phe188Asp + Gly201Gln + Val202Glu Ser98Glu + Gly127Asp + Ser131Glu + Val202Ala + Thr214Ser Gly99Asn + Gly127Glu + Ser131Glu + Gly156Gln + Asn211Glu Val94Asp + Pro128Asp + Ser131Asp + Tyr205Thr + Leu216Ser Gly101Glu + Gly126Ser + Gly127Asp + Tyr205His + Ser215Asp Ser160Asp + Tyr205Glu + Asn211Gln + Thr212Asp + Thr219Gly Gly62Asp + Gly127Gln + Gly153Pro + Ser187Glu + Leu216Glu Gly60Asp + Gly127Asp + Gly159Pro + Ser210Asp + Gly218Asn Asn61Ser + Gly99Asp + Thr102Asp + Ser203Asp + Thr212Gln Gly126Pro + Ser160Asp + Tyr205Met + Thr212Glu + Ser215Asp Thr65Ser + Gly127Asp + Thr132Ser + Thr212Asp + Ser215Asp Asn154Ser + Ser187Glu + Thr212Glu + Tyr213Gln + Ser215Glu Ser129Asp + Gly153Glu + Ser158Glu + Gly165Asn + Leu216Thr Ile106His + Leu125Pro + Ser129Glu + Ala186Glu + Ser203Glu Gly60Pro + Gly62Gln + Tyr205Thr + Ser215Glu + Thr219Glu Asp59Glu + Val94Pro + Gly130Gln + Ser187Asp + Thr219Asp Gly153Ser + Ser155Glu + Ser160Asp + Asn162Ser + Thr212Pro Ser155Asp + Gln161Glu + Ala186Thr + Thr214Gly + Ser215Glu Ser104Asp + Gly130Asn + Ser187Glu + Thr212Gln + Asn217Glu Ala105Asn + Ser203Glu + Thr212Glu + Asn217Ser + Thr219Asp Leu125His + Gly165Gln + Phe188Asp + Ser203Glu + Tyr205Leu Asn96Asp + Ser104Asp + Gly201Pro + Val202His + Leu216Ser Gly62Asp + Tyr103Val + Ser190Glu + Asn211Glu + Thr219Ser Gly62Asp + Ser190Glu + Val202Ser + Asn211Glu + Thr214Gln Ser100Asp + Pro128Asp + Tyr205Ile + Thr212Gln + Leu216His Thr132Gly + Gly156Asn + Gly159Glu + Ser190Asp + Ser203Glu Asn96Ser + Ser98Asp + Gly159Asp + Ser190Asp + Leu216Met Val94Asp + Ser98Glu + Thr132Asn + Phe188Cys + Thr219Asp Gly101Ser + Tyr103Glu + Gly153Glu + Tyr166His + Ser187Asp Gly99Ser + Ser160Asp + Ser190Glu + Val202Glu + Tyr205Ser Asn61Asp + Gly99Gln + Ala186Glu + Thr212Gln + Leu216Asp Ala186His + Phe188Gln + Ser190Glu + Pro200Ser + Asn217Asp Asn61Ser + Thr65Asp + Ser129Glu + Tyr205Glu + Tyr213Gly Asn61Asp + Gly62Ser + Ser203Asp + Asn211Gln + Tyr213Glu Thr102Asn + Gly130Asp + Tyr166Ser + Tyr205Asp + Gly218Asp Asn61Glu + Asn154Gln + Gln161Asn + Tyr205Asp + Gly218Glu Gly99Glu + Gly127Asp + Thr163Pro + Tyr213Ile + Asn217Glu Gly101Gln + Gly126Asp + Ser131Asp + Gly156Asn + Ser203Glu Thr102Asp + Gly130Glu + Pro209Asn + Thr212Ser + Ser215Asp Thr58Ser + Ser100Glu + Ser104Glu + Ile164Leu + Ser215Glu Asp59Glu + Ile106Val + Gly127Glu + Tyr166Asn + Ser190Glu Gly99Ser + Pro128Asp + Gly156Asp + Tyr205Cys + Asn217Asp Thr65Asp + Leu95Glu + Gly99Pro + Ser158Asp + Tyr205Gln Ser97Asp + Ala105His + Ser155Glu + Tyr205Asn + Pro209Glu Gly62Asn + Thr65Asp + Ser97Glu + Ser131Glu + Leu216Gly Gly130Pro + Gly153Glu + Asn154Gln + Gln161Asp + Pro209Asn Gly159Ser + Tyr166Glu + Tyr205Asp + Thr212Pro + Thr219Glu Gly62Asp + Leu95Cys + Ser100Glu + Thr163Pro + Ser187Asp Thr58Gly + Gly159Glu + Ser187Asp + Ser203Glu + Thr219Asn Thr58Pro + Tyr166Asp + Val202Met + Pro209Asp + Ser215Asp Leu95Asp + Thr102Pro + Ala105Gly + Gly127Glu + Leu216Glu Pro128Glu + Gln161Asp + Phe188Gln + Ser203Glu + Asn217Gln Leu125Pro + Pro128Glu + Thr132Asn + Gln161Glu + Leu216Asp Ser97Glu + Thr102Asp + Val202Thr + Ser215Glu + Gly218Ser Asp59Glu + Ser100Asp + Phe188Gln + Thr212Ser + Ser215Glu Gly101Gln + Thr102Asn + Gly130Glu + Thr163Glu + Ser210Asp Asn61Glu + Gly101Asn + Ser129Glu + Tyr205Thr + Leu216Glu Leu95Glu + Ile164Val + Thr212Glu + Tyr213Gly + Leu216Asp Thr58Gln + Gly126Glu + Gly156Gln + Thr212Asp + Leu216Asp Leu125Met + Gly126Ser + Ser131Asp + Thr212Asp + Leu216Asp Gly60Asn + Asn96Gln + Ser104Glu + Thr212Asp + Leu216Glu Thr65Asp + Gly126Gln + Ser158Asp + Gly165Asp + Leu216Val Ser158Asp + Gly165Glu + Ser203Asp + Leu216Ser + Gly218Gln Gly101Asp + Leu125Gly + Pro128Glu + Ala186Gly + Thr219Gln Thr65Glu + Asn96Glu + Tyr103Val + Ser155Glu + Met198Ala Thr65Asp + Asn96Glu + Leu125His + Asn162Glu + Tyr166His Asn61Ser + Ser100Asp + Phe188Asp + Ser203Glu + Gly218Ser Ser104Glu + Gly156Gln + Phe188Asp + Ser203Glu + Thr212Gly Gly99Asp + Tyr205Asp + Pro209Asp + Thr212Pro + Tyr213Met Thr102Asp + Asn154Ser + Tyr166Glu + Ser203Asp + Gly218Ser Gly101Glu + Ser129Asp + Ser157Asp + Thr214Ser + Leu216Asn Asp59Glu + Gly101Asp + Gly127Ser + Gly159Pro + Gln161Glu Gly60Asn + Gly127Asp + Thr132Asp + Asn162Gln + Ser203Glu Thr58Asn + Gly60Asp + Gly130Glu + Val202Ser + Thr212Glu Ser100Asp + Tyr166Asp + Ala186His + Tyr205His + Leu216Thr Ser97Asp + Gly101Ser + Ala105Glu + Tyr205Met + Ser215Glu Ser97Glu + Ala105Glu + Leu125Ala + Ser158Asp + Tyr205Cys Ser100Glu + Ile106His + Gly127Ser + Ser158Glu + Tyr166Glu Thr102Asn + Gly130Ser + Ser131Asp + Asn211Asp + Ser215Glu Gly127Gln + Ser131Glu + Asn211Asp + Tyr213His + Ser215Glu Ser129Glu + Ser158Glu + Val202Gly + Ser215Asp + Leu216Gln Leu125Pro + Pro128Ser + Ser157Glu + Ser203Glu + Gly218Ser Thr65Gln + Gln161Glu + Ser210Glu + Thr212Gln + Ser215Glu Val94Met + Gly101Glu + Ala105Asn + Ala186His + Thr212Asp

TABLE 36 Multi-loop Sextuple Mutation Variants Asn 61Ser + Gly 64Gln + Gly101Asp + Thr132Gly + Gly165Gln + Tyr205Pro Asn 96Ser + Gly101Ser + Ala105Gly + Asn154Glu + Tyr166Thr + Tyr205His Val 94A1a + Leu 9SThr + Gly126Gln + Pro209Ser + Ser215Asp + Leu216Gly Thr G5Gln + Leu125Asn + Asn162Asp + Tyr205Gln + Pro209Gly + Leu216Met Thr163Gln + Tyr205Cys + Thr212Pro + Leu216Met + Asn217Gln + Gly218Gln Gly 60Gln + Val 94Cys + Asn 96Ser + Gly127Pro + Gly159Asp + Asn162Ser Gly 99Ser + Gly126Asn + Gly127Pro + Gly153Asp + Gly159Pro + Ile164Ala Val 94Cys + Pro128Asp + Gly156Ser + Val202Thr + Tyr205Ala + Leu216Ala Asn 61Gln + Ala105Thr + Gly153Gln + Gly156Gln + Tyr166Ile + Asn211Gln Gly 60Asn + Ser104Asp + Ala105Asn + Gln16lSer + Thr163Ser + Tyr205Ala Gly 64Ser + Val 94Asn + Gly101Asn + Leu125Asn + Gly130Asp + Leu216Cys Gly159Pro + Gly165Pro + Ala186Thr + Gly201Pro + Thr212Pro + Leu216Ser Gly 99Asn + Gly159Pro + Ser203Glu + Tyr205Leu + Asn211Gln + Thr212Ser Thr 58Gln + Leu125Pro + Thr132Asp + Gly153Asn + Tyr213Ala + Leu216Gly Ile106Ala + Asn154Glu + Asn162Gln + Met198Thr + Ala199Asn + Tyr205Asn Asp 59Glu + Gly159Ser + Gly165Asn + Phe188Thr + Pro200Gln + Asn217Gln Gly 99Ser + Gly126Asn + Thr132Gly + Ser187Asp + Val202Ser + Tyr205His Tyr103Gln + Gly130Pro + Ser155Glu + Gly159Asn + Tyr205Pro + Thr212Ser Gly 60Pro + Val 94Ser + Ser100Glu + Tyr103Leu + Tyr205Ser + Thr219Pro Thr 58Gly + Leu125Ala + Val202Cys + Tyr205Asn + Ser215Asp + Leu216Ala Val 94Thr + Ser104Glu + Gly130Pro + Thr212Pro + Leu216Asn + Gly218Ser Ser 97Glu + Thr132Gly + Pro200Gly + Tyr205Ala + Asn211Ser + Tyr213Gln Gly 62Asn + Leu 95Asp + Tyr166Val + Tyr205Cys + Asn211Ser + Leu216Val Gly156Asn + Gly165Pro + Gly201Asn + Tyr213Ala + Thr214Gln + Asn217Glu Gly 62Ser + Ser 98Asp + Gly126Ser + Tyr205Pro + Thr212Ser + Tyr213Thr Ala105Gln + Gly127Ser + Gln161Asp + Thr163Gly + Tyr205Cys + Leu216Pro Val 94Ala + Gly130Gln + Ala186His + Ser203Asp + Tyr205Cys + Thr214Gln Thr 65Gln + Leu125Gly + Ser187Asp + Phe188Gln + Val202Asn + Thr212Asn Thr 58Ser + Ile106Pro + Thr132Gly + Gln161Asn + Tyr205Asn + Thr212Asp Ser100Asp + Ala105Thr + Leu125Pro + Tyr205Ile + Thr214Gly + Leu216Cys Thr102Ser + Gly127Pro + Pro128Gly + Ser203Glu + Tyr208His + Tyr213Thr Thr102Ser + Gln161Glu + Asn162Asp + Thr163Gln + Ala186His + Phe188Ile Gly 62Pro + Pro128Asp + Ser129Asp + Gly156Asn + Tyr213Cys + Gly218Asn Thr 58Gln + Gly127Pro + Ala186Gln + Va1202Asp + Ser203Asp + Leu216Val Gly156Ser + Phe188Gly + Ser203Glu + Tyr213Gln + Ser215Asp + Leu216Asp Gly101Gln + Ala105His + Asn162Gln + Ala186Gly + Ser203Glu + Ser215Glu Thr 58Pro + Thr163Asn + Ser203Glu + Tyr205Ile + Ser215Glu + Leu216Gln Thr 65Asn + Gly127Glu + Gly156Asn + Tyr166Glu + Phe188Met + Thr212Pro Leu 95Asp + Ser100Glu + Gly101Gln + Ile106Gln + Ile164Leu + Val202Ala Tyr103Pro + Gly127Glu + Ser129Asp + Gly130Glu + Asn162Gln + Leu216His Gly 60Gln + Gly165Ser + Ser203Glu + Tyr205Asp + Thr214Glu + Ser215Asp Leu 95Asn + Leu125Pro + Ser203Asp + Tyr205Thr + Thr214Glu + Ser215Asp Gly 62Asn + Tyr103Ile + Leu125Cys + Gly126Glu + Gly153Asp + Tyr166Cys Gly 99Ser + Pro128Glu + Gly130Asp + Phe188Asn + Leu216Gly + Gly218Pro Asp 59Glu + Gly 60Asp + Thr 65Asp + Asn162Ser + Thr163Gln + Tyr166Gln Phe188Glu + Tyr205Thr + Thr212Asn + Tyr213Gly + Asn217Glu + Gly218Asp Gly 62Gln + Ser203Glu + Tyr205Ser + Thr212Ser + Ser215Asp + Gly218Asp Ser187Glu + Ser190Asp + Tyr205Val + Pro209Ser + Thr214Ser + Thr219Ser Thr 58Ser + Asp 59Glu + Asn 96Asp + Asn154Ser + Ala186His + Leu216Ser Thr 65Gly + Thr102Gly + Leu125Asp + Gly165Asp + Ala199Ser + Val202Thr Leu125Asn + Ser158Glu + Gly159Asp + Ser190Glu + Thr212Gly + Leu216Val Thr 58Pro + Asn 61Asp + Val 94Glu + Asn 96Asp + Gly126Ser + Gly165Pro Ala186Glu + Val202Glu + Tyr205Ala + Tyr213Val + Ser215Asp + Asn217Glu Tyr103Val + Ser203Glu + Tyr205Asp + Thr212Glu + Ser215Asp + Leu216Ile Gly 62Ser + Ile106Leu + Pro128Ser + Asn1S4Asp + Ser157Glu + Thr214Ser Val 94Asp + Gly 99Asp + Pro128Gln + Ala199Gln + Tyr205Gln + Leu216Ile Asn 61Gln + Thr102Gln + Gly126Asp + Ser155Glu + Ser190Asp + Val202Ser Leu125Asp + Gly126Asp + Ala186Ser + Tyr205Gln + Gly218Glu + Thr219Pro Asn 61Glu + Ser100Asp + Thr102Asn + Gly165Gln + Phe188Leu + Val202His Tyr103Gly + Tyr205Leu + Thr212Glu + Ser215Glu + Leu216Glu + Asn217Asp Gly 99Pro + Ser104Glu + Ile106Thr + Ser131Asp + Gly201Gln + Thr214Ser Leu 95Ile + Gly126Glu + Gly153Glu + Ser187Asp + Phe188Asp + Asn211Gln Gly 62Asn + Leu 95Ser + Gly153Ser + Tyr205Asp + Thr212Glu + Leu216Asp Ala105Gly + Ile106Cys + Ser155Asp + Tyr205Asp + Ser215Asp + Leu216Asp Tyr103Ala + Ser155Glu + Tyr166Pro + Ser203Glu + Tyr205Glu + Ser215Glu Pro128Gly + Asn162Glu + Gly165Asn + Ser203Glu + Tyr205Glu + Ser215Glu Gly 99Pro + Leu125Gln + Gly130Asn + Ala186Asp + Phe188Ser + Ser203Glu Ile106Asn + Gly156Glu + Gly159Asn + Phe188Asp + Ser215Glu + Gly218Asp Gly156Pro + Asn162Asp + Ser203Glu + Tyr205Gln + Ser215Asp + Asn217Glu Gly153Asn + Ser155Glu + Ser157Asp + Ser190Glu + Thr212Asp + Leu216Ile Leu 95Glu + Gly 99Glu + Leu125Glu + Pro128Gly + Asn154Glu + Thr219Gly Gly 60Asp + Ile106Met + Asn162Ser + Ser203Glu + Tyr205Asp + Leu216Asp Ser 97Asp + Asn162Ser + Thr163Gln + Ser203Glu + Tyr205Asp + Leu216Asp Leu125Ile + Gly159Glu + Ser203Glu + Thr214Glu + Ser215Glu + Leu216Gln Gly156Ser + Ser158Glu + Tyr205Asp + Tyr213Glu + Ser215Asp + Leu216Cys Gly 62Gln + Asn154Asp + Ser155Asp + Ser160Asp + Gly165Glu + Tyr205Met Gly 64Pro + Ala186Glu + Ser187Asp + Ser203Glu + Tyr205His + Ser215Glu Tyr103Asp + Tyr166Ser + Ala186Asn + Val202Glu + Ser203Asp + Tyr205Glu Leu 95Gln + Gly126Gln + Gly127Asp + Pro128Ser + Ser155Asp + Ser158Glu Asp 59Glu + Ile106His + Tyr205Ala + Tyr213Glu + Ser215Glu + Leu216Asp Ser158Asp + Ser160Glu + Thr163Asp + Pro209Gln + Thr212Ser + Ser215Glu Gly 62Asp + Tyr103Met + Val202Ser + Asn211Glu + Thr214Gln + Leu216Ala Asp 59Glu + Ser 97Asp + Ser 98Asp + Gly153Ser + Gln161Asn + Ser203Glu Leu125Asn + Gly153Glu + Ser157Asp + Thr163Glu + Val202Asp + Thr214Pro Leu 95Glu + Ser100Asp + Leu125Asp + Pro128Asn + Ser131Asp + Tyr205Gly Gly126Gln + Ser158Glu + Ser203Asp + Ser215Glu + Leu216Thr + Gly218Asp Gly101Ser + Pro128Asp + Ser158Glu + Gly159Asp + Ala186Gly + Phe188Val Ser187Glu + Ser203Asp + Tyr205Cys + Pro209Gln + Ser215Asp + Leu216Ser Gly 62Ser + Ser187Glu + Met198Gln + Ser203Glu + Ser215Asp + Leu216His Thr132Asn + Gly165Pro + Ser187Glu + Ser203Glu + Tyr205His + Leu216Asp Asn 96Ser + Ser131Glu + Gly159Asp + Ser160Asp + Thr214Gln + Leu216Gly Gly130Glu + Ser158Asp + Gly159Glu + Thr163Gln + Tyr205Thr + Thr207Asn Gly130Asp + Ser131Glu + Gly165Pro + Ser190Glu + Val202Gly + Asn217Ser Leu 95Thr + Gly130Glu + Ser131Glu + Tyr205Ala + Ser210Glu + Gly218Gln Thr 58Gly + Ala105Glu + Ile106Gly + Tyr205Val + Ser215Glu + Leu216Asp Thr 58Asp + Asp 59Glu + Leu125Asn + Ser203Glu + Tyr205Cys + Thr212Ser Thr 58Glu + Asp 59Glu + Tyr103Asn + Pro209Gln + Thr212Asn + Ser215Glu Thr 65Ser + Ser 97Asp + Ser 98Glu + Ser104Glu + Thr163Gln + Leu216Ser Ser 97Glu + Ser 98Glu + Gly101Gln + Thr212Asn + Leu216His + Gly218Glu Gly101Asp + Thr102Glu + Ile106Val + Thr163Pro + Tyr205Glu + Ser215Asp Gly 60Pro + Ser129Glu + Val202Glu + Thr214Asp + Ser215Asp + Leu216Ile Gly 62Asp + Gly 99Glu + Tyr205Asn + Asn211Asp + Tyr213Met + Thr214Asp Thr 58Gly + Gly127Glu + Ser131Glu + Ser155Asp + Gly165Pro + Tyr205Cys Gly159Asp + Tyr166Gly + Tyr205Asp + Tyr213Asn + Ser215Asp + Leu216Thr Gly 62Glu + Gly101Ser + Thr132Gly + Ser203Glu + Asn211Asp + Tyr213Glu Asn 96Asp + Ser 97Asp + Leu125Pro + Gly127Gln + Ser203Asp + Ser215Asp Asp 59Glu + Val 94Glu + Ser 98Asp + Ser104Glu + Leu125Asn + Tyr213Gly Ser155Asp + Gly156Gln + Ser190Asp + Tyr205Glu + Thr212Ser + Leu216Ala Val 94Met + Gly 99Pro + Ile106Thr + Ser155Asp + Ser190Asp + Thr212Glu Gln161Ser + Thr163Glu + Val202Ala + Ser203Glu + Asn211Ser + Ser215Asp Gly156Glu + Ala186Asn + Ser203Asp + Thr212Gln + Ser215Glu + Leu216Gln Ser 98Glu + Ser203Asp + Val204Cys + Ser215Asp + Asn217Ser + Thr219Gln Asn 61Gln + Thr165Ser + Ser131Asp + Ser203Asp + Ser215Glu + Leu216Cys Ile106Val + Thr132Ser + Ser158Glu + Ser203Glu + Thr214Ser + Ser215Glu Val 94Thr + Gly153Glu + Ala186Gln + Ser203Glu + Ser215Asp + Thr219Gly Val 91Met + Thr102Asp + Pro128Glu + Ser131Asp + Gly165Asn + Thr219Ser Ser155Asp + Gln161Asp + Ser190Glu + Ser203Glu + Thr212Ser + Leu216Cys Asn 61Glu + Ser100Asp + Thr132Pro + Ala186Gly + Ser210Glu + Thr212Glu Ala105Glu + Thr132Ser + Gly201Ser + Ser203Glu + Thr212Pro + Asn217Asp Thr 58Gly + Gly 62Pro + Ala105Asp + Ser203Asp + Tyr208Ile + Leu216Glu Asn 96Asp + Gly 99Asp + Ile106Gly + Asn154Gln + Gln16lAsn + Thr214Glu Asn 96Glu + Gly 99Glu + Thr102Asn + Gly127Ser + Ser203Glu + Asn217Gln Asp 59Glu + Gly127Asp + Ser129Asp + Tyr213His + Leu216Val + Thr219Gly Thr 65Gln + Tyr205Ser + Pro209Gln + Ser210Glu + Thr212Glu + Thr219Asp Thr 58Asp + Asn 96Ser + Ser158Glu + Gln161Asp + Gly201Gln + Tyr205Val Asp 59Glu + Asn 61Glu + Tyr166His + Phe188Val + Thr212Gly + Ser215Asp Thr102Glu + Gly126Pr6 + Gly127Asn + Val202Asn + Pro209Asp + Asn211Glu Asn 61Gln + Gly159Gln + Ser187Asp + Pro209Asp + Asn211Asp + Gly218Ser Ser 97Asp + Ser 98Glu + Gly156Asp + Ser158Glu + Thr163Gln + Pro209Gln Gly 60Gln + Gly130Asn + Ser155Glu + Phe188Glu + Tyr205Asn + Ser215Glu Val 94Asp + Ile106Glu + Pro128Asp + Ser131Asp + Tyr205Thr + Leu216Ser Gly 60Pro + Ser 98Asp + Ser100Glu + Pro128Gly + Ser157Asp + Gly159Asp Thr 58Asn + Gly 99Asp + Thr102Gln + Pro209Asp + Thr212Glu + Tyr213Met Thr 65Glu + Tyr103Gln + Gly156Pr6 + Tyr166Asn + Phe188Tyr + Ser215Glu Val 94Asp + Gly101Glu + Ile164Gly + Ala186Pro + Ser203Asp + Leu216Pro Gly 60Asn + Asn 61Glu + Gly153Ser + Pro209Asp + Asn211Asp + Ser215Glu Thr132Gly + Ser157Glu + Ser187Asp + Ser203Asp + Tyr205Met + Ser215Glu Asn 61Asp + Asn 96Glu + Gly156Asn + Ser203Glu + Thr212Gln + Ser215Glu Gly 99Pro + Ser158Glu + Ser160Glu + Tyr205His + Thr212Ser + Ser215Glu Gly101Pro + Ser157Glu + Ser203Asp + Tyr205Asp + Thr212Ser + Leu216Cys Val 94A1a + Pro128Ser + Gly156Gln + Ser190Asp + Ser203Asp + Tyr205Glu Ile106Met + Leu125Ile + Ser160Asp + Ser203Asp + Tyr205Glu + Leu216Ser Leu 95Glu + Ser 97Glu + Thr102Glu + Gly159Asp + Tyr205Leu + Leu216Ala Leu125Cys + Gly126Ser + Gly159Asp + Gln161Glu + Ser215Glu + Asn217Glu Ser100Glu + Thr102Gly + Ala105Thr + Ser155Glu + Ser157Asp + Asn162Ser Ser 98Glu + Gly127Asn + Ser157Asp + Gln161Asn + Asn162Asp + Gly165Glu Asn 61Gln + Ser100Asp + Ser104Glu + Ile106Asp + Ser215Asp + Leu216Ala Ser131Glu + Gly159Asn + Val202Cys + Ser215Asp + Leu216Asp + Thr219Glu Leu125Cys + Gln161Asp + Asn162Glu + Ser203Glu + Leu216Thr + Gly218Glu Gly101Asp + Ala105Asp + Gly126Asn + Gln161Ser + Ala186Pro + Thr214Glu Ser100Glu + Thr102Glu + Tyr103Thr + Gly126Asp + Ser160Glu + Leu216Met Gly127Gln + Asn154Ser + Ser157Glu + Thr163Glu + Ser215Glu + Asn217Glu Leu 95I1e + Asn 96Glu + Thr102Gly + Tyr103Glu + Gly126Glu + Gly165Glu Ser129Asp + Ser131Asp + Thr132Asn + Gly159Ser + Tyr213Gln + Thr214Glu Gly101Pro + Gly126Glu + Ser129Asp + Tyr205Glu + Ser215Glu + Leu216Gly Leu 95Met + Pro128Gly + Ser129Glu + Gly153Glu + Thr163Gly + Leu216Ser Gly 60Glu + Val202Met + Tyr205Ile + Thr212Glu + Ser215Glu + Leu216Asp Thr 58Ser + Ser158Asp + Gln161Asn + Thr163Asp + Leu216Asp + Gly218Asp Ile106Ser + Ser158Asp + Ser187Asp + Ser190Asp + Tyr205Met + Ser215Asp Ser187Glu + Val202Gln + Tyr205Asn + Thr214Glu + Leu216Glu + Gly218Ser Asp 59Glu + Ser100Asp + Gly126Pro + Gly127Asp + Tyr166Glu + Thr212Asn Asp 59Glu + Gly 62Pro + Gly127Asn + Ala186Pro + Val202Glu + Pro209Asp Asp 59Glu + Asn 96Glu + Gly101Glu + Thr102Gln + Pro128Ser + Leu216Glu Gly159Asp + Asn162Asp + Ala186Gln + Val202Gln + Ser203Glu + Ser215Asp Gly159Ser + Ala186Asn + Ser210Asp + Tyr213Pro + Ser215Asp + Asn217Asp Ser104Glu + Ser131Asp + Thr132Gln + Asn162Ser + Gly165Asp + Tyr208Cys Gly156Glu + Ser158Asp + Thr163Gly + Ser203Asp + Thr212Asn + Leu216Ser Gly 64Pro + Thr 65Glu + Ala105Asp + Val202Thr + Ser215Asp + Leu216Glu Thr 58Pro + Gly 99Ser + Ser100Asp + Gly101Ser + Thr102Asp + Asn211Glu Gly 62Glu + Asn 96Ser + Ser100Asp + Gly101Ser + Ser210Asp + Tyr213Ala Gly 60Pro + Gln161Asp + Gly165Asp + Phe188Val + Ser190Glu + Ser210Glu Gly153Gln + Ser155Asp + Gly159Asn + Pro209Glu + Thr212Asp + Ser215Asp Thr 58Gln + Thr102Glu + Ser129Glu + Gly130Asp + Tyr205Ala + Ser215Asp Tyr103Ser + Asn154Asp + Ser187Glu + Val202Asn + Ser203Glu + Leu216Ser Leu125Pro + Pro128Ser + Ser157Glu + Gln161Glu + Ser203Glu + Gly218Ser Ser 97Glu + Gly 99Asp + Ala105Ser + Leu125Asp + Gly165Gln + Tyr205Asp Thr 65Asp + Leu 95Cys + Ser 97Asp + Asn162Ser + Thr212Glu + Ser215Asp Ser 97Glu + Ser100Glu + Gly156Asn + Ser157Glu + Ser160Glu + Tyr205Pro Leu 95Asp + Ala105Asp + Ser157Glu + Val202His + Tyr205Ile + Leu216Met Leu 95His + Gly156Pro + Ser157Asp + Asn162Glu + Ser187Glu + Leu216Asp Ser100Glu + Leu125Gly + Gly127Glu + Ser203Asp + Tyr208Val + Leu216Asp Asn154Ser + Gly165Asp + Ser190Glu + Thr212Gly + Ser215Asp + Gly218Pro Thr 58Gln + Ser 98Glu + Gly153Asp + Val202Ser + Tyr205Asn + Gly218Asp Asn 61Gln + Thr132Gly + Gly156Glu + Ser158Glu + Ser215Asp + Thr219Glu Val 94Glu + Ser 97Glu + Ala105Asp + Gly165Pro + Leu216Met + Thr219Asp Val 94Glu + Ser 97Glu + Ala105Asp + Asn154Gln + Gln161Asp + Ala186Asn Gly130Gln + Ser155Glu + Ser158Glu + Ala186Gln + Ser203Asp + Leu216cys Asn 61Gln + Ser160Asp + Ser203Glu + Val204Gln + Thr212Glu + Ser215Glu Thr102Glu + Ala105Asp + Ser131Glu + Ser203Glu + Tyr205Ala + Thr214Asn Thr 58Pro + Ser155Glu + Gly159Asn + Pro209Glu + Thr212Glu + Gly218Glu Asp 59Glu + Gly 99Asp + Gly126Ser + Met198Thr + Thr212Asp + Gly218Pro Thr132Ser + Asn154Ser + Ser157Asp + Val202Glu + Ser215Glu + Leu216Gly Val 94Asp + Gly 99Asn + Ser100Asp + Gly139Ser + Asn154Gln + Ser215Asp Pro128Glu + Gly156Ser + Ser160Glu + Ser187Asp + Ser190Glu + Thr212Ser Asn 61Gln + Ser 98Glu + Ile106cys + Ser203Asp + Tyr205Gly + Gly218Asp Ala105His + Ser158Glu + Ser187Glu + Ser203Glu + Tyr205Leu + Ser215Glu Asn 96Ser + Gly 99Gln + Gly153Glu + Ala186Glu + Tyr205Glu + Ser215Glu Asp 59Glu + Ser 98Glu + Gly153Glu + Ser155Glu + Phe188Pro + Leu216His Asn162Asp + Gly165Asp + Val204Gln + Tyr205Thr + Ser210Asp + Thr212Pro Gly101Pro + Gly156Asp + Tyr166Asp + Ser190Glu + Tyr205Asp + Thr212Asn Gly126Glu + Thr132Asp + Ser155Asp + Ser157Asp + Val202Gly + Tyr213Ser Gly 60Asp + Asn 61Asp + Asn154Glu + Ser158Glu + Gly165Gln + Tyr213Gln Gly 62Glu + Leu125His + Ser131Asp + Ser215Asp + Tyr166Leu + Ser203Glu Asp 59Glu + Val 94Asp + Thr102Gly + Ser203Glu + Tyr205Asp + Thr219Gly Ser104Glu + Tyr166Cys + Val202Thr + Thr212Asp + Ser215Asp + Asn217Glu Ser100Asp + Gly126Pro + Asn154Asp + Gly201Ser + Ser203Glu + Thr219Asp Gly101Asp + Pro128Asp + Ser155Asp + Ser187Asp + Thr207Gly + Thr212Gln Ile106Ser + Ser160Glu + Ser190Asp + Ser203Asp + Ser215Glu + Leu216Val Gly 99Gln + Gly153Asp + Ser160Asp + Phe188Leu + Ser190Asp + Ser210Asp Ser158Asp + Gly165Asn + Ser190Glu + Val202His + Tyr205Asp + Tyr213Leu Thr 65Asp + Leu 95Thr + Ser131Glu + Val202Ser + Ser203Asp + Leu216Glu Asn 61Glu + Gly165Ser + Met198Gln + Val202Glu + Tyr205Glu + Leu216Asn Val 94A1a + Gly126Asp + Asn154Glu + Tyr166Pro + Val202Gln + Thr212Glu Ser 97Glu + Leu125Glu + Ser155Glu + Gly159Asn + Tyr166Asp + Leu216Val Asn 96Glu + Ser 97Asp + Thr102Gln + Leu125Asp + Leu216Glu + Thr219Gln Tyr103Cys + Ser160Glu + Thr163Asp + Tyr208Ser + Leu216Met + Gly218Glu Val 94Cys + Leu125Glu + Ser155Asp + Gly156Asp + Gly201Gln + Ser215Glu Asn 61Ser + Thr 65Glu + Val 94Asp + Tyr103Pro + Ser215Asp + Asn217Asp Gly 99Gln + Gly127Asp + Thr132Pro + Ser155Glu + Ser187Glu + Ser203Asp Ser 98Glu + Ser129Glu + Gly153Glu + Ser190Asp + Tyr213Val + Thr214Pro Asn 61Ser + Gly130Glu + Phe188Glu + Tyr205Asp + Thr212Pro + Asn217Asp Ser158Glu + Gly159Glu + Val202Ser + Tyr205Met + Pro209Glu + Ser215Glu Asn 61Asp + Gly 99Asn + Gly101Asp + Ser203Asp + Tyr205Asp + Thr212Pro Ser 98Asp + Ser100Asp + Ser104Glu + Gly153Glu + Phe188Gly + Gly218Asn Leu 95Val + Gly 99Asp + Tyr103Val + Ala105Asp + Tyr205Asp + Ser215Asp Asp 59Glu + Asn 61Asp + Ser160Glu + Pro200Asn + Asn211Ser + Thr212Asp Gly 62Glu + Thr 65Gly + Ser 97Asp + Thr102Pro + Ser131Glu + Leu216Asp Gly126Pro + Pro128Glu + Gly156Glu + Ser158Asp + Tyr205Asn + Asn217Asp Asp 59Glu + Gly130Asn + Ser155Asp + Ser210Asp + Tyr213Gly + Gly218Asn Asn 96Gln + Gly 99Glu + Ser100Asp + Pro128Asp + Tyr205Cys + Asn217Glu Pro128Gly + Ser129Asp + Asn162Glu + Gly165Asn + Ser203Asp + Ser215Asp Asn 96Ser + Ser100Glu + Tyr103Glu + Tyr205His + Ser215Glu + Leu216Pro Asn 96Glu + Ser 98Glu + Ala186Asp + Val202Asn + Tyr213Met + Leu216Glu Gly 99Asp + Ser158Asp + Asn162Asp + Thr163Asn + Ser187Glu + Ala199Gly Leu 95Ser + Ser 97Asp + Ser 98Glu + Gly165Glu + Leu216Ile + Gly218Glu Gly101Asp + Leu125Asp + Gln161Asn + Tyr205Asp + Thr212Gly + Leu216Asn Ser160Asp + Val202His + Ser203Asp + Thr212Asp + Tyr213Val + Leu216Asp Leu 95Val + Tyr103Glu + Ala105Glu + Thr163Ser + Thr212Asp + Leu216Glu Ser129Asp + Gly130Ser + Ser157Asp + Gly165Asp + Tyr205Glu + Leu216Gln Thr132Asn + Gly156Glu + Tyr166Thr + Ser203Glu + Thr212Glu + Asn217Glu Asn 61Gln + Ser 97Glu + Ser187Glu + Ser203Glu + Pro209Gln + Ser215Glu Gly101Gln + Ala105Asp + Ser187Glu + Ser203Asp + Ser215Glu + Leu216His Gly 99Asn + Ser104Asp + Thr132Gly + Ser187Asp + Ser203Glu + Ser215Asp Gly 62Asp + Asn 96Gln + Ser104Glu + Tyr205Gln + Thr212Asp + Tyr213Val Val 94Gly + Asn 96Asp + Ala105Glu + Asn162Asp + Asn211Gln + Thr212Gln Ser 97Glu + Ser187Asp + Ser203Glu + Tyr213Gln + Leu216Glu + Asn217Gln Gly 64Pro + Ser104Glu + Gly126Glu + Tyr166Ser + Tyr205Glu + Ser215Glu Gly130Asn + Thr132Asp + Ser187Glu + Pro209Gln + Thr214Glu + Ser215Glu Gly126Glu + Ser160Asp + Tyr166Met + Val202Thr + Thr214Asp + Ser215Glu Ser 98Asp + Gly101Ser + Gly130Asp + Ser158Glu + Gly159Asp + Leu216Ala Thr 58Glu + Tyr103Pro + Gly130Glu + Ser131Asp + Ser158Glu + Ala199Pro Gly 60Asp + Asn 61Asp + Ser131Asp + Gly153Ser + Thr163Glu + Val202Cys Gly 99Asp + Pro128Asn + Asn162Glu + Thr212Ser + Leu216Glu + Asn217Glu Gly 60Ser + Ser 98Asp + Asn154Asp + Ser155Glu + Gly159Pro + Asn211Glu Tyr103Pro + Ser104Glu + Leu125Cys + Val202Glu + Ser203Glu + Pro209Glu Gly101Gln + Ser104Asp + Leu125Val + Val202Asp + Ser203Asp + Thr212Asp Thr 65Asn + Ser 98Asp + Gly159Asn + Gly165Asp + Ser215Asp + Leu216Asp Gly127Glu + Gly165Pro + Tyr205Cys + Ser210Asp + Ser215Glu + Leu216Glu Val 94Ser + Ser 98Glu + Gly 99Asp + Gly156Asp + Phe188Leu + Ser203Glu Gly 62Asp + Gly101Glu + Ser160Glu + Gln161Asp + Tyr205Met + Thr212Pro Ser 97Asp + Ser 98Glu + Ala105His + Ser157Glu + Thr212Ser + Ser215Asp Gly 62Gln + Ser 97Glu + Ser 98Asp + Gly126Glu + Gly130Pro + Thr212Glu Val 94Glu + Ser 97Asp + Leu125Asn + Thr163Ser + Ser210Asp + Leu216Ile Leu 95Glu + Asn 96Gln + Gly159Glu + Tyr205Glu + Pro209Ser + Ser215Glu Ser129Glu + Asn154Ser + Thr212Glu + Ser215Asp + Gly218Asp + Thr219Ser Asp 59Glu + Gly127Gln + Val202Glu + Asn211Glu + Thr212Ser + Ser215Asp Ser104Asp + Ala105Pro + Ser187Glu + Ser203Glu + Tyr205Asp + Tyr213Pro Thr 58Pro + Asn 96Asp + Gly127Glu + Ser155Glu + Asn162Ser + Thr212Ser Thr 58Gly + Leu125Asn + Gly156Asp + Gln161Glu + Tyr205Gly + Ser215Asp Gly 60Ser + Gly156Asp + Pro209Ser + Thr214Asp + Leu216Thr + Thr219Asp Leu125His + Gly127Gln + Ser131Glu + Ser203Asp + Asn211Asp + Ser215Glu Thr 58Glu + Pro128Glu + Gly165Gln + Ala186His + Ser203Glu + Ser215Glu Ser131Asp + Ser158Glu + Ser203Asp + Tyr205His + Ser215Asp + Leu216Gln Thr 65Asn + Gly130Asp + Ser158Asp + Ser203Asp + Pro209Ser + Ser215Glu Gly 60Gln + Thr102Glu + Ser157Asp + Ser203Asp + Ser215Asp + Thr219Gln Gly 64Gln + Gly127Asp + Ser160Glu + Ser203Asp + Ser215Glu + Gly218Gln Ile106Leu + Thr132Asp + Thr163Asp + Ser203Glu + Ser215Asp + Leu216Ile Gly126Ser + Ser160Asp + Thr212Glu + Ser215Glu + Leu216Va1 + Thr219Ser Gly 60Pro + Gly101Asn + Ser158Glu + Tyr205Ile + Thr212Asp + Ser215Glu Gly 62Glu + Gly 99Ser + Val202Ser + Tyr205Asp + Thr212Asn + Thr214Ser Val 94His + Thr132Glu + Gly165Asp + Tyr166cys + Ser203Asp + Leu216Glu Gly 62Asn + Ser131Asp + Ser157Glu + Gln161Asn + Ser203Glu + Leu216Glu Gly 60Pro + Val 94Asp + Ala105Glu + Gly127Pro + Gln161Asp + Ser210Asp Gly 62Gln + Ser 97Asp + Asn154Asp + Ser190Asp + Ser203Glu + Thr212Ser Ser 97Glu + Asn154Glu + Gly159Asn + Ser190Asp + Tyr205Thr + Asn211Glu Thr 65Glu + Ser 98Asp + Thr102Pro + Gly126Asp + Gly165Asp + Gly218Gln Asn 96Glu + Tyr103Asp + Gly159Asp + Asn162Asp + Tyr208Leu + Thr212Pro Gly 62Asp + Ser104Glu + Ile106Asp + Ser157Asp + Gly218Ser + Thr219Pro Asn 96Glu + Gly 99Asp + Ser160Asp + Gly201Ser + Asn211Gln + Ser215Glu Ile106Pro + Gly153Asp + Gly165Glu + Tyr166Ser + Ser203Asp + Thr212Asp Ser 97Asp + Gly 99Asn + Ser158Asp + Ser210Glu + Thr212Glu + Leu216Thr Asp 59Glu + Ser 98Asp + Ser160Glu + Gly201Ser + Thr212Gly + Tyr213Asn Val 94Ser + Ser 97Asp + Ser187Glu + Ser190Asp + Pro209Asp + Leu216Asn Gly101Gln + Gly126Asp + Ser131Asp + Asn154Glu + Gly156Asn + Ser203Asp Gly 64Ser + Ser 98Glu + Ser100Glu + Ala186Glu + Thr212Glu + Leu216Pro Ser 97Asp + Gly 99Asp + Val202Asp + Thr207Asn + Asn211Glu + Leu216Met Thr 65Pro + Thr132Gly + Ala186Asp + Val202Ala + Ser203Glu + Thr212Asp Ile106Asp + Gly127Ser + Asn154Glu + Gly156Glu + Thr214Pro + Ser215Glu Thr 58Asp + Ser129Glu + Thr132Asn + Gly165Asp + Leu216Val + Gly218Asp Thr 58Pro + Gly 60Glu + Gly 62Glu + Thr102Asp/ + Ser158Glu + Leu216Ala Pro128Asp + Asn154Glu + Phe188Val + Val202Cys + Ser203Asp + Tyr205Asn Gly101Gln + Ala105His + Ser155Asp + Tyr166Val + Ala186Asp + Pro209Glu Leu 95Va1 + Ser155Glu + Tyr166Ala + Ala186Glu + Ser210Asp + Leu216Met Asn 96Asp + Gly101Glu + Asn162Glu + Val202Thr + Ser210Asp + Leu216Asn Ser129Glu + Ser157Asp + Phe188Ala + Ser190Glu + Val202Ser + Tyr205Glu Ser131Asp + Ser158Asp + Ser160Asp + Tyr205Ile + Ser215Asp + Leu216Met Gly 62Glu + Leu 95Glu + Tyr103Pro + Ala186His + Ser203Glu + Thr207Gly Ser100Glu + Leu125Ala + Ser157Glu + Ser203Asp + Tyr205Asp + Leu216Cys Thr102Gln + Ser104Asp + Gly153Pro + Ser158Glu + ‘Ser203Glu + Tyr205Asp Asn 61Glu + Asn 96Gln + Ser 98Asp + Ser158Asp + Thr212Asn + Thr219Asp Val 94Thr + Thr132Asp + Ser155Asp + Ser157Glu + Thr207Gly + Ser215Asp Pro128Gly + Gly130Pro + Ser158Glu + Ser187Glu + Phe188Cys + Ser203Asp Thr 65Glu + Gly130Pro + Gly159Glu + Gly165Asp + Phe188Met + Pro209Asp Thr163Glu + Val202Asp + Tyr205Cys + Thr212Asp + Leu216Pro + Gly218Glu Ser 97Glu + Gly101Asn + Gly130Glu + Asn154Asp + Thr163Gly + Gly218Glu Thr 65Gly + Ala105Glu + Ser155Asp + Gln161Glu + Phe188Val + Asn217Gln Thr 65Gln + Tyr103Asp + Pro128Glu + Ser155Asp + Ser215Asp + Leu216Ser Thr132Glu + Ala186Gln + Ser167Glu + Val202Pro + Thr207Gly + Asn217Asp Gly 60Glu + Ser129Glu + Ser131Glu + Gly165Asn + Ser203Glu + Pro209Gln Gly101Pro + Pro128Asp + Gly130Asp + Ser203Glu + Tyr205Ile + Thr212Asp Val 94Glu + Asn 96Glu + Asn154Asp + Tyr205Ala + Thr212Glu + Leu216Cys Ser 97Glu + Thr102Ser + Ser157Asp + Ser215Asp + Leu216Ser + Asn217Glu Ser 98Asp + Gly156Glu + Ser158Glu + Gln161Ser + Ser203Asp + Gly218Asn Gly130Ser + Gly156Asp + Ser158Glu + Ser210Asp + Thr212Gln + Ser215Asp Ser100Glu + Thr102Asp + Gly126Pro + Gly127Ser + Ser187Glu + Ser215Glu Asn 96Glu + Gly127Ser + Pro128Gln + Ser190Asp + Tyr213Asp + Ser215Glu Pro128Glu + Gly153Ser + Ser158Glu + Gly165Ser + Ala186Gly + Ser187Glu Thr 65Glu + Gly153Asp + Thr163Asp + Gly165Asn + Met198His + Ser203Glu Leu125Gly + Ser129Glu + Thr163Glu + Ala186His + Ser203Glu + Tyr205Leu Thr 58Glu + Asn 61Ser + Thr 65Gly + Ser158Asp + Asn162Asp + Ser203Glu Thr 65Asn + Val 94Thr + Gly126Glu + Ser157Asp + Asn162Glu + Leu216Asp Asp 59Glu + Ser 97Glu + Pro128Asn + Thr132Glu + Ser157Asp + Tyr205Gly Thr 58Glu + Gly 60Gln + Leu 95Glu + Val202Gln + Tyr205Gln + Ser215Asp Asp 59Glu + Ser157Asp + Asn162Asp + Tyr166Asn + Leu216Ile + Asn217Glu Ser157Glu + Asn162Glu + Phe188Gly + Ser203Asp + Asn211Asp + Thr212Ser Ser157Glu + Asn162Asp + Ser210Asp + Thr212Ser + Tyr213Leu + Leu216Asp Gly 60Pro + Ser 97Glu + Ser100Asp + Ala105His + Ser203Glu + Asn211Asp Asn 61Asp + Leu125His + Gly156Asn + Pro200Gln + Thr212Glu + Leu216Asp Thr 65Asp + Ser 97Asp + Leu125Asn + Ser157Glu + Thr163Gly + Thr212Asp Gly 62Glu + Thr 65Asn + Asn162Glu + Thr163Ser + Asn211Asp + Ser215Glu Val 94Glu + Asn 96Ser + Gly101Pro + Thr102Asp + Ser155Glu + Thr212Asp Gly 60Glu + Ser 98Glu + Gly 99Gln + Ser104Glu + Ala105Thr + Thr214Asp Ser155Asp + Ser187Asp + Ala199Gln + Ser203Asp + Tyr205Val + Thr212Glu Ser104Glu + Ile106Cys + Thr163Ser + Gly165Glu + Ser190Glu + Thr212Asp Val 94Thr + Ile106Asp + Gly130Asn + Ser131Asp + Ser203Glu + Tyr205Pro Ser 98Glu + Pro128Glu + Ser155Asp + Ser160Glu + Ala186Ser + Pro209Asn Thr 58Glu + Gly159Ser + Tyr186Glu + Ser190Glu + Thr212Pro + Leu216Ser Leu 95Glu + Leu125His + Gly126Asp + Tyr205Gln + Ser215Asp + Thr219Asn Asp 59Glu + Gly 99Asp + Ala105Glu + Ala186Pro + Ser187Asp + Thr212Pro Asn 96Gln + Ser 98Glu + Gly104Pro + Thr132Glu + Val202Asp + Ser215Asp Gly 99Asp + Pro128Asp + Thr132Asn + Val202Asp + Ser215Asp + Leu216His Val 94Gln + Gly101Asp + Ser104Asp + Thr163Pro + Ser187Asp + Ser215Asp Leu 95Met + Gly101Asp + Ser104Glu + Gly153Glu + Tyr205Ala + Ser215Glu Gly101Glu + Ser104Glu + Ser203Asp + Tyr205Ile + Ser210Glu + Thr212Pro Val 94Glu + Ser100Glu + Ile100Met + Gly130Glu + Val202Glu + Thr214Asn Ser 98Asp + Ile106Asp + Asn154Asp + Gly165Asp + Val202Met + Leu216Gln Leu 95Met + Thr102Ser + Gly127Asn + Ser190Glu + Val202Gly + Asn217Asp Gly 62Pro + Ile106Gln + Gly126Asp + Ser129Glu + Ser160Asp + Ser215Asp Gly 62Asp + Asn154Glu + Ser160Glu + Ala186Thr + Ser187Asp + Leu216Thr Gly 62Ser + Thr 65Ser + Ser155Asp + Gly159Glu + Ser203Glu + Thr219Gln

II. Cleaning Compositions

In another embodiment of the present invention, an effective amount of one or more of the enzyme variants are included in compositions useful for cleaning a variety of surfaces in need of proteinaceous stain removal. Such cleaning compositions include detergent compositions for cleaning hard surfaces, unlimited in form (e.g., liquid and granular); detergent compositions for cleaning fabrics, unlimited in form (e.g., granular, liquid and bar formulations); dishwashing compositions (unlimited in form); oral cleaning compositions, unlimited in form (e.g., dentifrice, toothpaste and mouthwash formulations); denture cleaning compositions, unlimited in form (e.g., liquid, tablet); and contact lens cleaning compositions, unlimited in form (e.g., liquid, tablet).

The cleaning compositions also comprise, in addition to the Subtilisin DY variants described hereinbefore, one or more cleaning composition materials compatible with the protease enzyme. the term “cleaning composition material”, as used herein, means any liquid, solid or gaseous material selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, granule, bar, spray, stick, paste, gel), which materials are also compatible with the Subtilisin DY variant used in the composition. The specific selection of cleaning composition materials are readily made by considering the surface material to be cleaned, the desired form of the composition for the cleaning condition during use (e.g., through the wash detergent use). The term “compatible”, as used herein, means the cleaning composition materials do not reduce the proteolytic activity of the Subtilisin DY variant to such an extent that the protease is not effective as desired during normal use situations. Specific cleaning composition materials are exemplified in detail hereinafter.

As used herein, “effective amount of enzyme variant” refers to the quantity of enzyme variant necessary to achieve the enzymatic activity necessary in the specific cleaning composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and is based on many factors, such as the particular enzyme variant used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar) composition is required, and the like. Preferably the cleaning compositions comprise from about 0.0001% to about 10% of one or more enzyme variants of the present invention, more preferably from about 0.001% to about 1%, more preferably still from about 0.01% to about 0.1%. Several examples of various cleaning compositions wherein the enzyme variants may be employed are discussed in further detail below. All parts, percentages and ratios used herein are by weight unless otherwise specified.

As used herein, “non-fabric cleaning compositions” include hard surface cleaning compositions, dishwashing compositions, oral cleaning compositions, denture cleaning compositions and contact lens cleaning compositions.

A. Cleaning Compositions for Hard Surfaces, Dishes and Fabrics.

The enzyme variants of the present invention can be used in a variety of detergent compositions where high sudsing and good insoluble substrate removal are desired. Thus the enzyme variants can be used with various conventional ingredients to provide fully-formulated hard-surface cleaners, dishwashing compositions, fabric laundering compositions and the like. Such compositions can be in the form of liquids, granules, bars and the like. Such compositions can be formulated as modem “concentrated” detergents which contain as much as 30%-60% by weight of surfactants.

The cleaning compositions herein can optionally, and preferably, contain various anionic, nonionic, zwitterionic, etc., surfactants. Such surfactants are typically present at levels of from about 5% to about 35% of the compositions.

Nonlimiting examples of surfactants useful herein include the conventional C₁₁-C₁₈ alkyl benzene sulfonates and primary and random alkyl sulfates, the C₁₀-C₁₈ secondary (2,3) alkyl sulfates of the formulas CH₃(CH₂)x(CHOSO₃)⁻M⁺)CH₃ and CH₃(CH₂)y(CHOSO₃ ⁻M⁺) CH₂CH₃ wherein x and (y+1) are integers of at least about 7, preferably at least about 9, and M is a water-solubilizing cation, especially sodium, the C₁₀-C₁₈ alkyl alkoxy sulfates (especially EO 1-5 ethoxy sulfates), C₁₀-C₁₈ alkyl alkoxy carboxylates (especially the EO 1-5 ethoxycarboxylates), the C₁₀-C₁₈ alkyl polyglycosides, and their corresponding sulfated polyglycosides, C₁₂-C₁₈ alpha-sulfonated fatty acid esters, C₁₂-C₁₈ alkyl and alkyl phenol alkoxylates (especially ethoxylates and mixed ethoxy/propoxy), C₁₂-C₁₈ betaines and sulfobetaines (“sultaines”), C₁₀-C₁₈ amine oxides, and the like. The alkyl alkoxy sulfates (AES) and alkyl alkoxy carboxylates (AEC) are preferred herein. (Use of such surfactants in combination with the aforesaid amine oxide andlor betaine or sultaine surfactants is also preferred, depending on the desires of the formulator.) Other conventional useful surfactants are listed in standard texts. Particularly useful surfactants include the C₁₀-C₁₈ N-methyl glucamides disclosed in U.S. Pat. No. 5,194,639, Connor et al., issued Mar. 16, 1993, incorporated herein by reference.

A wide variety of other ingredients useful in detergent cleaning compositions can be included in the compositions herein, including other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, solvents for liquid formulations, etc. If an additional increment of sudsing is desired, suds boosters such as the C₁₀-C₁₆ alkolamides can be incorporated into the compositions, typically at about 1% to about 10% levels. The C₁₀-C₁₄ monoethanol and diethanol amides illustrate a typical class of such suds boosters. Use of such suds boosters with high sudsing adjunct surfactants such as the amine oxides, betaines and sultaines noted above is also advantageous. If desired, soluble magnesium salts such as MgCl₂, MgSO₄, and the like, can be added at levels of, typically, from about 0.1% to about 2%, to provide additionally sudsing.

The liquid detergent compositions herein can contain water and other solvents as carriers. Low molecular weight primary or secondary alcohols exemplified by methanol, ethanol, propanol, and isopropanol are suitable. Monohydric alcohols are preferred for solubilizing surfactants, but polyols such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (e.g., 1,3-propanediol, ethylene glycol, glycerine, and 1,2-propanediol) can also be used. The compositions may contain from about 5% to about 90%, typically from about 10% to about 50% of such carriers.

The detergent compositions herein will preferably be formulated such that during use in aqueous cleaning operations, the wash water will have a pH between about 6.8 and about 11.0. Finished products thus are typically formulated at this range. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.

When formulating the hard surface cleaning compositions and fabric cleaning compositions of the present invention, the formulator may wish to employ various builders at levels from about 5% to about 50% by weight. Typical builders include the 1-10 micron zeolites, polycarboxylates such as citrate and oxydisuccinates, layered silicates, phosphates, and the like. Other conventional builders are listed in standard formularies.

Likewise, the formulator may wish to employ various additional enzymes, such as cellulases, lipases, amylases and proteases in such compositions, typically at levels of from about 0.001% to about 1% by weight. Various detersive and fabric care enzymes are well-known in the laundry detergent art.

Various bleaching compounds, such as the percarbonates, perborates and the like, can be used in such compositions, typically at levels from about 1% to about 15% by weight. If desired, such compositions can also contain bleach activators such as tetraacetyl ethylenediamine, nonanoyloxybenzene sulfonate, and the like, which are also known in the art. Usage levels typically range from about 1% to about 10% by weight.

Various soil release agents, especially of the anionic oligoester type, various chelating agents, especially the aminophosphonates and ethylenediaminedisuccinates, various clay soil removal agents, especially ethoxylated tetraethylene pentamine, various dispersing agents, especially polyacrylates and polyasparatates, various brighteners, especially anionic brighteners, various suds suppressors, especially silicones and secondary alcohols, various fabric softeners, especially smectite clays, and the like can all be used in such compositions at levels ranging from about 1% to about 35% by weight. Standard formularies and published patents contain multiple, detailed descriptions of such conventional materials.

Enzyme stabilizers may also be used in the cleaning compositions. Such enzyme stabilizers include propylene glycol (preferably from about 1% to about 10%), sodium formate (preferably from about 0.1% to about 1%) and calcium formate (preferably from about 0.1% to about 1%).

1. Hard Surface Cleaning Compositions

As used herein “hard surface cleaning composition” refers to liquid and granular detergent compositions for cleaning hard surfaces such as floors, walls, bathroom tile, and the like. Hard surface cleaning compositions of the present invention comprise an effective amount of one or more enzyme variants of the present invention, preferably from about 0.001% to about 10%, more preferably from about 0.01% to about 5%, more preferably still from about 0.05% to about 1% by weight of active enzyme of the composition. In addition to comprising one or more of the enzyme variants, such hard surface cleaning compositions typically comprise a surfactant and a water-soluble sequestering builder. In certain specialized products such as spray window cleaners, however, the surfactants are sometimes not used since they may produce a filmy/streaky residue on the glass surface.

The surfactant component, when present, may comprise as little as 0.1% of the compositions herein, but typically the compositions will contain from about 0.25% to about 10%, more preferably from about 1% to about 5% of surfactant.

Typically the compositions will contain from about 0.5% to about 50% of a detergency builder, preferably from about 1% to about 10%.

Preferably the pH should be in the range of about 8 to 12. Conventional pH adjustment agents such as sodium hydroxide, sodium carbonate or hydrochloric acid can be used if adjustment is necessary.

Solvents may be included in the compositions. Useful solvents include, but are not limited to, glycol ethers such as diethyleneglycol monohexyl ether, diethyleneglycol monobutyl ether, ethyleneglycol monobutyl ether, ethyleneglycol monohexyl ether, propyleneglycol monobutyl ether, dipropyleneglycol monobutyl ether, and diols such as 2,2,4-trimethyl-1,3-pentanediol and 2-ethyl-1,3-hexanediol. When used, such solvents are typically present at levels of from about 0.5% to about 15%, preferably from about 3% to about 11%.

Additionally, highly volatile solvents such as isopropanol or ethanol can be used in the present compositions to facilitate faster evaporation of the composition from surfaces when the surface is not rinsed after “full strength” application of the composition to the surface. When used, volatile solvents are typically present at levels of from about 2% to about 12% in the compositions.

The hard surface cleaning composition embodiment of the present invention is illustrated by the following examples.

EXAMPLES 7-12

Liquid Hard Surface Cleaning Compositions Example No. Component 7 8 9 10 11 12 Phe188Asp 0.05 0.50 0.02 0.03 0.10 0.03 Val202Ala — — — — 0.20 0.02 Na₂DIDA* EDTA** — — 2.90 2.90 — — Na Citrate — — — — 2.90 2.90 NaC₁₂ Alkyl-benzene 1.95 — 1.95 — 1.95 — sulfonate NaC₁₂ Alkylsulfate — 2.20 — 2.20 — 2.20 NaC₁₂(ethoxy)*** — 2.20 — 2.20 — 2.20 sulfate C₁₂ Dimethylamine — 0.50 — 0.50 — 0.50 oxide Na Cumene sulfonate 1.30 — 1.30 — 1.30 — Hexyl Carbitol*** 6.30 6.30 6.30 6.30 6.30 6.30 Water**** balance to 100% *Disodium N-diethyleneglycol-N,N-iminodiacetate **Na₄ ethylenediamine diacetic acid ***Diethyleneglycol monohexyl ether ****All formulas adjusted to pH 7

In Examples 7-10, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Phe188Asp, with substantially similar results.

In Examples 11-12, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Phe188Asp and Val202Ala, with substantially similar results.

EXAMPLES 13-18

Spray Compositions for Cleaning Hard Surfaces and Removing Household Mildew Example No. Component 13 14 15 16 17 18 Asn96Asp + Tyr103His 0.50 0.05 0.60 0.30 0.20 0.30 Pro200Gly + Gly201Ser + — — — — 0.30 0.10 Ser210Asp Sodium octyl sulfate 2.00 2.00 2.00 2.00 2.00 2.00 Sodium dodecyl sulfate 4.00 4.00 4.00 4.00 4.00 4.00 Sodium hydroxide 0.80 0.80 0.80 0.80 0.80 0.80 Silicate (Na) 0.04 0.04 0.04 0.04 0.04 0.04 Perfume 0.35 0.35 0.35 0.35 0.35 0.35 Water balance to 100%

Product pH is about 7.

In Examples 13-16, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Asn 96Asp+Tyr103His, with substantially similar results.

In Examples 17-18, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Asn 96Asp+Tyr103His and Pro200Gly+Gly201Ser+Ser210Asp, with substantially similar results.

2. Dishwashing Compositions

In another embodiment of the present invention, dishwashing compositions comprise one or more enzyme variants of the present invention. As used herein, “dishwashing composition” refers to all forms for compositions for cleaning dishes, including but not limited to, granular and liquid forms. The dishwashing composition embodiment of the present invention is illustrated by the following examples.

EXAMPLES 19-24

Dishwashing Composition Example No. Component 19 20 21 22 23 24 Gly127Asn 0.05 0.50 0.02 0.40 0.10 0.03 Gly201Asn + Tyr205Gly + — — — — 0.40 0.02 Thr207Gln + Tyr213Ile C₁₂—C₁₄ N-methyl- 0.90 0.90 0.90 0.90 0.90 0.90 glucamide C₁₂ ethoxy (1) sulfate 12.00 12.00 12.00 12.00 12.00 12.00 2-methyl undecanoic acid 4.50 4.50 4.50 4.50 4.50 4.50 C₁₂ ethoxy (2) carboxylate 4.50 4.50 4.50 4.50 4.50 4.50 C₁₂ alcohol ethoxylate (4) 3.00 3.00 3.00 3.00 3.00 3.00 C₁₂ amine oxide 3.00 3.00 3.00 3.00 3.00 3.00 Sodium cumene sulfonate 2.00 2.00 2.00 2.00 2.00 2.00 Ethanol 4.00 4.00 4.00 4.00 4.00 4.00 Mg⁺⁺ (as MgCl₂) 0.20 0.20 0.20 0.20 0.20 0.20 Ca⁺⁺ (as CaCl₂) 0.40 0.40 0.40 0.40 0.40 0.40 Water balance to 100%

Product pH is adjusted to 7.

In Examples 19-22, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly127Asn, with substantially similar results.

In Examples 23-24, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly127Asn and Gly201Asn+Tyr205Gly+Thr207Gln+Tyr213Ile, with substantially similar results.

3. Fabric Cleaning Compositions

In another embodiment of the present invention, fabric cleaning compositions comprise one or more enzyme variants of the present invention. As used herein, “fabric cleaning composition” refers to all forms for detergent compositions for cleaning fabrics, including but not limited to, granular, liquid and bar forms. Preferred fabric cleaning compositions are those in the liquid form.

a. Granular Fabric Cleaning Compositions

The granular fabric cleaning compositions of the present invention contain an effective amount of one or more enzyme variants of the present invention, preferably from about 0.001% to about 10%, more preferably from about 0.005% to about 5%, more preferably from about 0.01% to about 1% by weight of active enzyme of the composition. In addition to one or more enzyme variants, the granular fabric cleaning compositions typically comprise at least one surfactant, one or more builders, and, in some cases, a bleaching agent.

The granular fabric cleaning composition embodiment of the present invention is illustrated by the following examples.

EXAMPLES 25-28

Granular Fabric Cleaning Composition Example No. Component 25 26 27 28 Gly156Glu + Asn162Gln 0.10 0.20 0.03 0.05 Leu95Glu — — 0.02 0.05 C₁₃ linear alkyl benzene sulfonate 22.00 22.00 22.00 22.00 Phosphate (as sodium 23.00 23.00 23.00 23.00 tripolyphosphates) Sodium carbonate 23.00 23.00 23.00 23.00 Sodium silicate 14.00 14.00 14.00 14.00 Zeolite 8.20 8.20 8.20 8.20 Chelant (diethylaenetriamine- 0.40 0.40 0.40 0.40 pentaacetic acid) Sodium sulfate 5.50 5.50 5.50 5.50 Water balance to 100%

In Examples 25-26, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly156Glu+Asn162Gln, with substantially similar results.

In Examples 27-28, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly156Glu+Asn162Gln and Leu 95Glu, with substantially similar results.

EXAMPLES 29-32

Granular Fabric Cleaning Composition Example No. Component 29 30 31 32 Gln161Glu 0.10 0.20 0.03 0.05 Ala186Ser + Phe188Ala + Ser190Glu — — 0.02 0.05 C₁₂ alkyl benzene sulfonate 12.00 12.00 12.00 12.00 Zeolite A (1-10 micrometer) 26.00 26.00 26.00 26.00 2-butyl octanoic acid 4.00 4.00 4.00 4.00 C₁₂—C₁₄ secondary (2,3) alkyl sulfate, 5.00 5.00 5.00 5.00 Na salt Sodium citrate 5.00 5.00 5.00 5.00 Optical brightener 0.10 0.10 0.10 0.10 Sodium sulfate 17.00 17.00 17.00 17.00 Water and minors balance to 100%

In Examples 29-30, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gln161Glu, with substantially similar results.

In Examples 31-32, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gln161Glu and Ala186Ser+Phe188Ala+Ser190Glu, with substantially similar results.

EXAMPLES 33-36

Granular Fabric Cleaning Composition Example No. Component 33 34 35 36 Asn96Gln + Ile106Ala 0.10 0.20 0.03 0.05 Gly60Asn + Asn61Glu + Gly153Ser + — — 0.02 0.05 Pro209Asp + Asn211Asp + Ser215Glu C₁₃ linear alkyl benzene sulfonate 22.00 22.00 22.00 22.00 Phosphate (as sodium 23.00 23.00 23.00 23.00 tripolyphosphates) Sodium carbonate 23.00 23.00 23.00 23.00 Sodium silicate 14.00 14.00 14.00 14.00 Zeolite 8.20 8.20 8.20 8.20 Chelant (diethylaenetriamine- 0.40 0.40 0.40 0.40 pentaacetic acid) Sodium sulfate 5.50 5.50 5.50 5.50 Water balance to 100%

In Examples 33-34, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Asn 96Gln+Ile106Ala, with substantially similar results.

In Examples 35-36, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Asn 96Gln+Ile106Ala and Gly, 60Asn+Asn 61Glu+Gly153Ser+Pro209Asp+Asn211Asp+Ser215Glu, with substantially similar results.

EXAMPLES 37-40

Granular Fabric Cleaning Composition Example No. Component 37 38 39 40 Gly60Asn 0.10 0.20 0.03 0.05 Ala186Asn + Ser187Asp + Phe188Gln + — — 0.02 0.05 Ser190Glu C₁₂ alkyl benzene sulfonate 12.00 12.00 12.00 12.00 Zeolite A (1-10 micrometer) 26.00 26.00 26.00 26.00 2-butyl octanoic acid 4.00 4.00 4.00 4.00 C₁₂—C₁₄ secondary (2,3) alkyl sulfate, 5.00 5.00 5.00 5.00 Na salt Sodium citrate 5.00 5.00 5.00 5.00 Optical brightener 0.10 0.10 0.10 0.10 Sodium sulfate 17.00 17.00 17.00 17.00 Water and minors balance to 100%

In Examples 37-38, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly60Asn, with substantially similar results.

In Examples 39-40, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly60Asn and Ala186Asn+Ser187Asp+Phe188Gln+Ser190Glu, with substantially similar results.

EXAMPLES 41-42

Granular Fabric Cleaning Composition Example No. Component 41 42 Linear alkyl benzene sulphonate 11.4 10.70 Tallow alkyl sulphate 1.80 2.40 C₁₄₋₁₅ alkyl sulphate 3.00 3.10 C₁₄₋₁₅ alcohol 7 times ethoxylated 4.00 4.00 Tallow alcohol 11 times ethoxylated 1.80 1.80 Dispersant 0.07 0.1 Silicone fluid 0.80 0.80 Trisodium citrate 14.00 15.00 Citric acid 3.00 2.50 Zeolite 32.50 32.10 Maleic acid acrylic acid copolymer 5.00 5.00 Diethylene triamine penta methylene 1.00 0.20 phosphonic acid Ser215Asp 0.30 0.30 Lipase 0.36 0.40 Amylase 0.30 0.30 Sodium silicate 2.00 2.50 Sodium sulphate 3.50 5.20 Polyvinyl pyrrolidone 0.30 0.50 Perborate 0.5 1 Phenol sulphonate 0.1 0.2 Peroxidase 0.1 0.1 Minors Up to 100 Up to 100

EXAMPLES 43-44

Granular Fabric Cleaning Composition Example No. Component 43 44 Sodium linear C₁₂ alkyl benzene-sulfonate 6.5 8.0 Sodium sulfate 15.0 18.0 Zeolite A 26.0 22.0 Sodium nitrilotriacetate 5.0 5.0 Polyvinyl pyrrolidone 0.5 0.7 Tetraacetylethylene diamine 3.0 3.0 Boric acid 4.0 — Perborate 0.5 1 Phenol sulphonate 0.1 0.2 Val202Met + Thr212Pro 0.4 0.4 Fillers (e.g., silicates; carbonates; Up to 100 Up to 100 perfumes; water)

EXAMPLE 45

Compact Granular Fabric Cleaning Composition Component Weight % Alkyl Sulphate 8.0 Alkyl Ethoxy Sulphate 2.0 Mixture of C₂₅ and C₄₅ alcohol 3 and 7 times ethoxylated 6.0 Polyhydroxy fatty acid amide 2.5 Zeolite 17.0 Layered silicate/citrate 16.0 Carbonate 7.0 Maleic acid acrylic acid copolymer 5.0 Soil release polymer 0.4 Carboxymethyl cellulose 0.4 Poly (4-vinylpyridine) -N-oxide 0.1 Copolymer of vinylimidazole and vinylpyrrolidone 0.1 PEG2000 0.2 Thr65Glu + Ile106Met + Leu125His + Tyr205Leu + 0.5 Ser215Asp Lipase 0.2 Cellulase 0.2 Tetracetylethylene diamine 6.0 Percarbonate 22.0 Ethylene diamine disuccinic acid 0.3 Suds suppressor 3.5 Disodium-4,4′-bis (2-morpholino -4-anilino-s-triazin-6- 0.25 ylamino) stilbene-2,2′-disulphonate Disodium-4,4′-bis (2-sulfostyril) biphenyl 0.05 Water, Perfume and Minors Up to 100

EXAMPLE 46

Granular Fabric Cleaning Composition Component Weight % Linear alkyl benzene sulphonate 7.6 C₁₆—C₁₈ alkyl sulfate 1.3 C_(14-15 alcohol 7 times ethoxylated) 4.0 Coco-alkyl-dimethyl hydroxyethyl ammonium chloride 1.4 Dispersant 0.07 Silicone fluid 0.8 Trisodium citrate 5.0 Zeolite 4A 15.0 Maleic acid acrylic acid copolymer 4.0 Diethylene triamine penta methylene phosphonic acid 0.4 Perborate 15.0 Tetraacetylethylene diamine 5.0 Smectite clay 10.0 Poly (oxy ethylene) (MW 300,000) 0.3 Thr207Asn + Tyr208Gln + Thr212Ser + Tyr213Glu + 0.4 Ser215Glu Lipase 0.2 Amylase 0.3 Cellulase 0.2 Sodium silicate 3.0 Sodium carbonate 10.0 Carboxymethyl cellulose 0.2 Brighteners 0.2 Water, perfume and minors Up to 100

EXAMPLE 47

Granular Fabric Cleaning Composition Component Weight % Linear alkyl benzene sulfonate 6.92 Tallow alkyl sulfate 2.05 C₁₄₋₁₅ alcohol 7 times ethoxylated 4.4 C₁₂₋₁₅ alkyl ethoxy sulfate - 3 times ethoxylated 0.16 Zeolite 20.2 Citrate 5.5 Carbonate 15.4 Silicate 3.0 Maleic acid acrylic acid copolymer 4.0 Carboxymethyl cellulase 0.31 Soil release polymer 0.30 Gly 60Asp + Asn 61Asp + Gly 62Asp + Thr 65Gln 0.2 Lipase 0.36 Cellulase 0.13 Perborate tetrahydrate 11.64 Perborate monohydrate 8.7 Tetraacetylethylene diamine 5.0 Diethylene tramine penta methyl phosphonic acid 0.38 Magnesium sulfate 0.40 Brightener 0.19 Perfume, silicone, suds suppressors 0.85 Minors Up to 100

b. Liquid Fabric Cleaning Compositions

Liquid fabric cleaning compositions of the present invention comprise an effective amount of one or more enzyme variants of the present invention, preferably from about 0.005% to about 5%, more preferably from about 0.01% to about 1%, by weight of active enzyme of the composition. Such liquid fabric cleaning compositions typically additionally comprise an anionic surfactant, a fatty acid, a water-soluble detergency builder and water.

The liquid fabric cleaning composition embodiment of the present invention is illustrated by the following examples.

EXAMPLES 48-52

Liquid Fabric Cleaning Compositions Example No. Component 48 49 50 51 52 Val94Gly + Gly99Pro + Ile106Val 0.05 0.03 0.30 0.03 0.10 Gly218Pro — — — 0.01 0.20 C₁₂—C₁₄ alkyl sulfate, Na 20.00 20.00 20.00 20.00 20.00 2-butyl octanoic acid 5.00 5.00 5.00 5.00 5.00 Sodium citrate 1.00 1.00 1.00 1.00 1.00 C₁₀ alcohol ethoxylate (3) 13.00 13.00 13.00 13.00 13.00 Monethanolamine 2.50 2.50 2.50 2.50 2.50 Water/propylene glycol/ethanol balance to 100% (100:1:1)

In Examples 48-50 the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Val94Gly+Gly99Pro+Ile106Val, with substantially similar results.

In Examples 51-52, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Val94Gly+Gly99Pro+Ile106Val and Gly218Pro, with substantially similar results.

EXAMPLES 53-57

Liquid Fabric Cleaning Compositions Example No. Component 53 54 55 56 57 Gly156Ser 0.05 0.03 0.30 0.03 0.10 Ser187Glu — — — 0.01 0.20 C₁₂—C₁₄ alkyl sulfate, Na 20.00 20.00 20.00 20.00 20.00 2-butyl octanoic acid 5.00 5.00 5.00 5.00 5.00 Sodium citrate 1.00 1.00 1.00 1.00 1.00 C₁₀ alcohol ethoxylate (3) 13.00 13.00 13.00 13.00 13.00 Monethanolamine 2.50 2.50 2.50 2.50 2.50 Water/propylene glycol/ethanol balance to 100% (100:1:1)

In Examples 53-55 the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly156Ser, with substantially similar results.

In Examples 56-57, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly156Ser and Ser187Glu, with substantially similar results.

EXAMPLES 58-59

Liquid Fabric Cleaning Composition Example No. Component 58 59 C₁₂₋₁₄ alkenyl succinic acid 3.0 8.0 Citric acid monohydrate 10.0 15.0 Sodium C₁₂₋₁₅ alkyl sulphate 8.0 8.0 Sodium sulfate of C₁₂₋₁₅ alcohol 2 times ethoxylated — 3.0 C₁₂₋₁₅ alcohol 7 times ethoxylated — 8.0 C₁₂₋₁₅ alcohol 5 times ethoxylated 8.0 — Diethylene triamine penta (methylene phosphonic acid) 0.2 — Oleic acid 1.8 — Ethanol 4.0 4.0 Propanediol 2.0 2.0 Gly101Glu + Ser104Glu + Ser203Asp + Tyr205Ile + 0.2 0.2 Ser210Glu + Thr212Pro Polyvinyl pyrrolidone 1.0 2.0 Suds suppressor 0.15 0.15 NaOH up to pH 7.5 Perborate 0.5 1 Phenol sulphonate 0.1 0.2 Peroxidase 0.4 0.1 Waters and minors up to 100 parts

In each of Examples 58 and 59 herein, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly101Glu+Ser104Glu+Ser203Asp+Tyr205Ile+Ser210Glu+Thr212Pro, with substantially similar results.

EXAMPLES 60-62

Liquid Fabric Cleaning Composition Example No. Component 60 61 62 Citric Acid 7.10 3.00 3.00 Fatty Acid 2.00 — 2.00 Ethanol 1.93 3.20 3.20 Boric Acid 2.22 3.50 3.50 Monoethanolamine 0.71 1.09 1.09 1,2 Propanediol 7.89 8.00 8.00 NaCumene Sulfonate 1.80 3.00 3.00 NaFormate 0.08 0.08 0.08 NaOH 6.70 3.80 3.80 Silicon anti-foam agent 1.16 1.18 1.18 Asn61Glu + Gly64Ser + Thr65Gly 0.0145 — — Ser129Asp + Gly130Asn — 0.0145 — Leu216Asn — — 0.0145 Lipase 0.200 0.200 0.200 Cellulase — 7.50 7.50 Soil release polymer 0.29 0.15 0.15 Anti-foaming agents 0.06 0.085 0.085 Brightener 36 0.095 — — Brightener 3 — 0.05 0.05 C₁₂ alkyl benzenesulfonic acid 9.86 — — C₁₂₋₁₅ alkyl polyethoxylate (2.5) sulfate 13.80 18.00 18.00 C₁₂ glucose amide — 5.00 5.00 C₁₂₋₁₃ alkyl polyethoxylate (9) 2.00 2.00 2.00 Water, perfume and minors balance to 100%

c. Bar Fabric Cleaning Compositions

Bar fabric cleaning compositions of the present invention suitable for hand-washing soiled fabrics contain an effective amount of one or more enzyme variants of the present invention, preferably from about 0.001% to about 10%, more preferably from about 0.01% to about 1% by weight of the composition.

The bar fabric cleaning composition embodiment of the present invention is illustrated by the following examples.

EXAMPLES 63-66

Bar Fabric Cleaning Compositions Example No. Component 63 64 65 66 Val202Ser + Asn211Asp 0.3 — 0.1 0.02 Gly156Gln + Ile164Thr — — 0.4 0.03 C₁₂—C₁₆ alkyl sulfate, Na 20.0 20.0 20.0 20.00 C₁₂—C₁₄ N-methyl glucamide 5.0 5.0 5.0 5.00 C₁₁—C₁₃ alkyl benzene sulfonate, Na 10.0 10.0 10.0 10.00 Sodium carbonate 25.0 25.0 25.0 25.00 Sodium pyrophosphate 7.0 7.0 7.0 7.00 Sodium tripolyphosphate 7.0 7.0 7.0 7.00 Zeolite A (0.1-.10μ) 5.0 5.0 5.0 5.00 Carboxymethylcellulose 0.2 0.2 0.2 0.20 Polyacrylate (MW 1400) 0.2 0.2 0.2 0.20 Coconut monethanolamide 5.0 5.0 5.0 5.00 Brightener, perfume 0.2 0.2 0.2 0.20 CaSO₄ 1.0 1.0 1.0 1.00 MgSO₄ 1.0 1.0 1.0 1.00 Water 4.0 4.0 4.0 4.00 Filler* balance to 100% *Can be selected from convenient materials such as CaCO₃, talc, clay, silicates, and the like.

In Examples 63-64 the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Val202Ser+Asn211Asp, with substantially similar results.

In Examples 65-46, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Val202Ser+Asn211Asp and Gly156Gln+Ile164Thr, with substantially similar results.

EXAMPLES 67-70

Bar Fabric Cleaning Compositions Example No. Component 67 68 69 70 Val204Thr + Pro209Asp + Tyr213Cys 0.3 — 0.1 0.02 Gly126Gln + Thr214Gly + Ser215Glu — 0.3 0.4 0.03 C₁₂—C₁₆ alkyl sulfate, Na 20.0 20.0 20.0 20.00 C₁₂—C₁₄ N-methyl glucamide 5.0 5.0 5.0 5.00 C₁₁—C₁₃ alkyl benzene sulfonate, Na 10.0 10.0 10.0 10.00 Sodium carbonate 25.0 25.0 25.0 25.00 Sodium pyrophosphate 7.0 7.0 7.0 7.00 Sodium tripolyphosphate 7.0 7.0 7.0 7.00 Zeolite A (0.1-.10μ) 5.0 5.0 5.0 5.00 Carboxymethylcellulose 0.2 0.2 0.2 0.20 Polyacrylate (MW 1400) 0.2 0.2 0.2 0.20 Coconut monethanolamide 5.0 5.0 5.0 5.00 Brightener, perfume 0.2 0.2 0.2 0.20 CaSO₄ 1.0 1.0 1.0 1.00 MgSO₄ 1.0 1.0 1.0 1.00 Water 4.0 4.0 4.0 4.00 Filler* balance to 100% *Can be selected from convenient materials such as CaCO₃, talc, clay, silicates, and the like.

In Example 67, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Val204Thr+Pro209Asp+Tyr213Cys, with substantially similar results.

In Example 68, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly126Gln+Thr214Gly+Ser215Glu, with substantially similar results.

In Examples 69-70, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Val204Thr+Pro209Asp+Tyr213Cys and Gly126Gln+Thr214Gly+Ser215Glu, with substantially similar results.

B. Additional Cleaning Compositions

In addition to the hard surface cleaning, dishwashing and fabric cleaning compositions discussed above, one or more enzyme variants of the present invention may be incorporated into a variety of other cleaning compositions where hydrolysis of an insoluble substrate is desired. Such additional cleaning compositions include but are not limited to, oral cleaning compositions, denture cleaning compositions, and contact lens cleaning compositions.

1. Oral Cleaning Compositions

In another embodiment of the present invention, a pharmaceutically-acceptable amount of one or more enzyme variants of the present invention are included in compositions useful for removing proteinaceous stains from teeth or dentures. As used herein, “oral cleaning compositions” refers to dentifrices, toothpastes, toothgels, toothpowders, mouthwashes, mouth sprays, mouth gels, chewing gums, lozenges, sachets, tablets, biogels, prophylaxis pastes, dental treatment solutions, and the like. Preferably, the oral cleaning compositions comprise from about 0.0001% to about 20% of one or more enzyme variants of the present invention, more preferably from about 0.001% to about 10%, more preferably still from about 0.01% to about 5%, by weight of the composition, and a pharmaceutically-acceptable carrier. As used herein, “pharmaceutically-acceptable” means that drugs, medicaments or inert ingredients which the term describes are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, incompatibility, instability, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio.

Typically, the pharmaceutically-acceptable oral cleaning carrier components of the oral cleaning components of the oral cleaning compositions will generally comprise from about 50% to about 99.99%, preferably from about 65% to about 99.99%, more preferably from about 65% to about 99%, by weight of the composition.

The pharmaceutically-acceptable carrier components and optional components which may be included in the oral cleaning compositions of the present invention are well known to those skilled in the art. A wide variety of composition types, carrier components and optional components useful in the oral cleaning compositions are disclosed in U.S. Pat. No. 5,096,700, Seibel, issued Mar. 17, 1992; U.S. Pat. No. 5,028,414, Sampathkumar, issued Jul. 2, 1991; and U.S. Pat. No. 5,028,415, Benedict, Bush and Sunberg, issued Jul. 2, 1991; all of which are incorporated herein by reference.

The oral cleaning composition embodiment of the present invention is illustrated by the following examples.

EXAMPLES 71-74

Dentifrice Composition Example No. Component 71 72 73 74 Thr214Asp 2.000 3.500 1.500 2.000 Sorbitol (70% aqueous solution) 35.000 35.000 35.000 35.000 PEG-6* 1.000 1.000 1.000 1.000 Silica dental abrasive** 20.000 20.000 20.000 20.000 Sodium fluoride 0.243 0.243 0.243 0.243 Titanium dioxide 0.500 0.500 0.500 0.500 Sodium saccharin 0.286 0.286 0.286 0.286 Sodium alkyl sulfate (27.9% 4.000 4.000 4.000 4.000 aqueous solution) Flavor 1.040 1.040 1.040 1.040 Carboxyvinyl Polymer*** 0.300 0.300 0.300 0.300 Carrageenan**** 0.800 0.800 0.800 0.800 Water balance to 100% *PEG-6 = Polyethylene glycol having a molecular weight of 600. **Precipitated silica identified as Zeodent 119 offered by J. M. Huber. ***Carbopol offered by B. F. Goodrich Chemical Company. ****Iota Carrageenan offered by Hercules Chemical Company.

In Examples 71-74 the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Thr214Asp, with substantially similar results.

EXAMPLES 75-78

Mouthwash Composition Example No. Component 75 76 77 78 Thr132Glu 3.00 7.50 1.00 5.00 SDA 40 Alcohol 8.00 8.00 8.00 8.00 Flavor 0.08 0.08 0.08 0.08 Emulsifier 0.08 0.08 0.08 0.08 Sodium Fluoride 0.05 0.05 0.05 0.05 Glycerin 10.00 10.00 10.00 10.00 Sweetener 0.02 0.02 0.02 0.02 Benzoic acid 0.05 0.05 0.05 0.05 Sodium hydroxide 0.20 0.20 0.20 0.20 Dye 0.04 0.04 0.04 0.04 Water balance to 100%

In Examples 75-78, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Thr132Glu, with substantially similar results.

EXAMPLES 79-82

Lozenge Composition Example No. Component 79 80 81 82 Ser97Glu + Ile106Leu 0.01 0.03 0.10 0.02 Sorbitol 17.50 17.50 17.50 17.50 Mannitol 17.50 17.50 17.50 17.50 Starch 13.60 13.60 13.60 13.60 Sweetener 1.20 1.20 1.20 1.20 Flavor 11.70 11.70 11.70 11.70 Color 0.10 0.10 0.10 0.10 Corn Syrup balance to 100%

In Examples 79-82, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Ser 97Glu+Ile106Leu, with substantially similar results.

EXAMPLES 83-86

Chewing Gum Composition Example No. Component 83 84 85 86 Leu125Ala + Ser129Glu + Gly130Pro + 0.03 0.02 0.10 0.05 Ser131Asp Sorbitol crystals 38.44 38.40 38.40 38.40 Paloja-T gum base* 20.00 20.00 20.00 20.00 Sorbitol (70% aqueous solution) 22.00 22.00 22.00 22.00 Mannitol 10.00 10.00 10.00 10.00 Glycerine 7.56 7.56 7.56 7.56 Flavor 1.00 1.00 1.00 1.00 *Supplied by L. A. Dreyfus Company.

In Examples 83-86, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Leu125Ala+Ser129Glu+Gly130Pro+Ser131Asp, with substantially similar results.

2. Denture Cleaning Compositions

In another embodiment of the present invention, denture cleaning compositions for cleaning dentures outside of the oral cavity comprise one or more enzyme variants of the present invention. Such denture cleaning compositions comprise an effective amount of one or more of the enzyme variants, preferably from about 0.0001% to about 50% of one or more of the enzyme variants, more preferably from about 0.001% to about 35%, more preferably still from about 0.01% to about 20%, by weight of the composition, and a denture cleansing carrier. Various denture cleansing composition formats such as effervescent tablets and the like are well known in the art (see for example U.S. Pat. No. 5,055,305, Young, incorporated herein by reference), and are generally appropriate for incorporation of one or more of the enzyme variants for removing proteinaceous stains from dentures.

The denture cleaning composition embodiment of the present invention is illustrated by the following examples.

EXAMPLES 87-90

Two-layer Effervescent Denture Cleansing Tablet Example No. Component 87 88 89 90 Acidic Layer Thr212Pro + Thr219Pro 1.0 1.5 0.01 0.05 Tartaric acid 24.0 24.0 24.00 24.00 Sodium carbonate 4.0 4.0 4.00 4.00 Sulphamic acid 10.0 10.0 10.00 10.00 PEG 20,000 4.0 4.0 4.00 4.00 Sodium bicarbonate 24.5 24.5 24.50 24.50 Potassium persulfate 15.0 15.0 15.00 15.00 Sodium acid pyrophosphate 7.0 7.0 7.00 7.00 Pyrogenic silica 2.0 2.0 2.00 2.00 TAED* 7.0 7.0 7.00 7.00 Ricinoleylsulfosuccinate 0.5 0.5 0.50 0.50 Flavor 1.0 1.0 1.00 1.00 Alkaline Layer Sodium perborate monohydrate 32.0 32.0 32.00 32.00 Sodium bicarbonate 19.0 19.0 19.00 19.00 EDTA 3.0 3.0 3.00 3.00 Sodium tripolyphosphate 12.0 12.0 12.00 12.00 PEG 20,000 2.0 2.0 2.00 2.00 Potassium persulfate 26.0 26.0 26.00 26.00 Sodium carbonate 2.0 2.0 2.00 2.00 Pyrogenic silica 2.0 2.0 2.00 2.00 Dye/flavor 2.0 2.0 2.00 2.00 *Tetraacetylethylene diamine

In Examples 87-90, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Thr212Pro+Thr219Pro, with substantially similar results.

3. Contact Lens Cleaning Compositions

In another embodiment of the present invention, contact lens cleaning compositions comprise one or more enzyme variants of the present invention. Such contact lens cleaning compositions comprise an effective amount of one or more of the enzyme variants, preferably from about 0.01% to about 50% of one or more of the enzyme variants, more preferably from about 0.01% to about 20%, more preferably still from about 1% to about 5%, by weight of the composition, and a contact lens cleaning carrier. Various contact lens cleaning composition formats such as tablets, liquids and the like are well known in the art (see for example U.S. Pat. No. 4,863,627, Davies, Meaken and Rees, issued Sep. 5, 1989; U.S. Pat. No. Re. 32,672, Huth, Lam and Kirai, reissued May 24, 1988; U.S. Pat. No. 4,609,493, Schäfer, issued Sep. 2, 1986; U.S. Pat. No. 4,690,793, Ogunbiyi and Smith, issued Sep. 1, 1987; U.S. Pat. No. 4,614,549, Ogunbiyi, Riedhammer and Smith, issued Sep. 30, 1986; and U.S. Pat. No. 4,285,738, Ogata, issued Aug. 25, 1981; each of which are incorporated herein by reference), and are generally appropriate for incorporation of one or more enzyme variants of the present invention for removing proteinaceous stains from contact lens.

The contact lens cleaning composition embodiment of the present invention is illustrated by the following examples.

EXAMPLES 91-94

Enzymatic Contact Lens Cleaning Solution Example No. Component 91 92 93 94 Gly156Glu + Thr163Ser + Asn211Ser + 0.01 0.5 0.1 2.0 Thr212Asn Glucose 50.00 50.0 50.0 50.0 Nonionic surfactant (polyoxyethlene- 2.00 2.0 2.0 2.0 polyoxypropylene copolymer) Anionic surfactant (polyoxyethylene- 1.00 1.0 1.0 1.0 alkylphenylether sodium sulfricester) Sodium chloride 1.00 1.0 1.0 1.0 Borax 0.30 0.3 0.3 0.3 Water balance to 100%

In Examples 91-94, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly156Glu+Thr163Ser+Asn211Ser+Thr212Asn, with substantially similar results.

While particular embodiments of the subject invention have been described, it will be obvious to those skilled in the art that various changes and modifications of the subject invention can be made without departing from the spirit and scope of the invention. It is intended to cover, in the appended claims, all such modifications that are within the scope of the invention.

1 274 amino acids amino acid single linear 1 Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val 1 5 10 15 Gln Ala Gln Gly Tyr Lys Gly Ala Asn Val Lys Val Gly Ile Ile Asp 20 25 30 Thr Gly Ile Ala Ala Ser His Thr Asp Leu Lys Val Val Gly Gly Ala 35 40 45 Ser Phe Val Ser Gly Glu Ser Tyr Asn Thr Asp Gly Asn Gly His Gly 50 55 60 Thr His Val Ala Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val 65 70 75 80 Leu Gly Val Ala Pro Asn Val Ser Leu Tyr Ala Ile Lys Val Leu Asn 85 90 95 Ser Ser Gly Ser Gly Thr Tyr Ser Ala Ile Val Ser Gly Ile Glu Trp 100 105 110 Ala Thr Gln Asn Gly Leu Asp Val Ile Asn Met Ser Leu Gly Gly Pro 115 120 125 Ser Gly Ser Thr Ala Leu Lys Gln Ala Val Asp Lys Ala Tyr Ala Ser 130 135 140 Gly Ile Val Val Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Ser 145 150 155 160 Gln Asn Thr Ile Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val 165 170 175 Gly Ala Val Asp Ser Asn Lys Asn Arg Ala Ser Phe Ser Ser Val Gly 180 185 190 Ala Glu Leu Glu Val Met Ala Pro Gly Val Ser Val Tyr Ser Thr Tyr 195 200 205 Pro Ser Asn Thr Tyr Thr Ser Leu Asn Gly Thr Ser Met Ala Ser Pro 210 215 220 His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys Tyr Pro Thr Leu 225 230 235 240 Ser Ala Ser Gln Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Asn Leu 245 250 255 Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala 260 265 270 Ala Gln 

What is claimed is:
 1. A Subtilisin DY variant having a modified amino acid sequence of the Subtilisin DY wild-type amino acid sequence set forth in SEQ ID NO:1, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region, a fifth loop region and a sixth loop region; wherein the modified amino acid sequence comprises a substitution at one or more positions in one of the loop regions; wherein A. when the substitution occurs in the first loop region, the substitution occurs at one of positions 60, 64 or 65; wherein a. when a substitution occurs at position 60, the substituting amino acid is Asn, Gln, Pro or Ser; b. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and c. when a substitution occurs at position 65, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; B. when the substitution occurs in the second loop region, the substitution occurs at one of positions 94, 95, 96, 99 or 105; wherein a. when a substitution occurs at position 94, the substituting amino acid is Ala, Gln, Gly, His, Met, Pro or Ser; b. when a substitution occurs at position 95, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; c. when a substitution occurs at position 96, the substituting amino acid is Gln or Ser; d. when a substitution occurs at position 99, the substituting amino acid is Asn, Gln, Pro or Ser; and e. when a substitution occurs at position 105, the substituting amino acid is Asn, Gln, Gly, His, Pro, Ser or Thr; C. when the substitution occurs in the third loop region, the substitution occurs at one of positions 128,131 or 132; wherein a. when a substitution occurs at position 128, the substituting amino acid is Asn, Gln or Ser; b. when a substitution occurs at position 131, the substituting amino acid is Asp or Glu; and c. when a substitution occurs at position 132, the substituting amino acid is Asn, Gln, Gly, Pro or Ser; D. when the substitution occurs in the fourth loop region, the substitution occurs at position 154 wherein the substituting amino acid is Ser; E. when a substitution occurs in the fifth loop region, the substitution occurs at position 188; wherein the substituting amino acid is Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val; and F. when the substitution occurs in the sixth loop region, the substitution occurs at one of positions 199, 200, 201, 202, 204, 205, 206, 207, 208, 209, 211, 212, 213, or 219; wherein a. when a substitution occurs at position 199, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; b. when a substitution occurs at position 200, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; c. when a substitution occurs at position 201, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 202, the substituting amino acid is Ala, Cys, Gln, Gly, His, Met, Pro or Ser; e. when a substitution occurs at position 204, the substituting amino acid is Ala, Cys, Gln, Gly, His, Met, Pro or Ser; f. when a substitution occurs at position 205, the substituting amino acid is Ala, Asn, Gln, Gly, His, Ile, Leu, Met, Pro or Ser; g. when a substitution occurs at position 206, the substituting amino acid is Asp or Glu; h. when a substitution occurs at position 207, the substituting amino acid is Asn, Gln, Gly, Pro or Ser; i. when a substitution occurs at position 208, the substituting amino acid is Ala, Asn, Cys, Gln, Gly, His, Ile, Met, Pro or Ser; j. when a substitution occurs at position 209, the substituting amino acid is Asn, Gln, Gly, or Ser; k. when a substitution occurs at position 211, the substituting amino acid is Gln or Ser; l. when a substitution occurs at position 212, the substituting amino acid is Asn, Gln, Gly, Pro or Ser; m. when a substitution occurs at position 213, the substituting amino acid is Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Met or Pro; and n. when a substitution occurs at position 219, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; whereby the Subtilisin DY variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type Subtilisin DY and wherein an amino acid substitution at the subtilisin DY positions 94 to 103, 106, 125 to 128, 153 to 166, 188, 203, 212 to 214 and 216 is combined with at least one further substitution at a corresponding position selected from subtilisin DY positions 59, 60, 64, 65, 104, 105, 129, 131, 132, 187, 189, 190,199 to 202, 204, 206, 209 to 211, 215 and
 219. 2. The Subtilisin DY variant of claim 1, wherein the substitution occurs in the first loop region.
 3. The Subtilisin DY variant of claim 1, wherein the substitution occurs in the second loop region.
 4. The Subtilisin DY variant of claim 1, wherein the substitution occurs in the third loop region.
 5. The Subtilisin DY variant of claim 1, wherein the substitution occurs in the fourth loop region.
 6. The Subtilisin DY variant of claim 1, wherein the substitution occurs in the fifth loop region.
 7. The Subtilisin DY variant of claim 1, wherein one or more substitutions occur in the sixth loop region.
 8. A Subtilisin DY variant having a modified amino acid sequence of the Subtilisin DY wild-type amino acid sequence as set forth in SEQ ID NO:1, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region, a fifth loop region and a sixth loop region; wherein the modified amino acid sequence comprises a substitution at two or more positions in one or more of the loop regions; wherein A. when a substitution occurs in the first loop region, the substitution occurs at one or more of positions 58, 59, 60, 61, 62, 64 or 65; wherein a. when a substitution occurs at position 58, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; b. when a substitution occurs at position 59, the substituting amino acid is Glu; c. when a substitution occurs at position 60, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 61, the substituting amino acid is Asp, Gln, Glu or Ser; e. when a substitution occurs at position 62, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; f. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and g. when a substitution occurs at position 65, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; B. when a substitution occurs in the second loop region, the substitution occurs at one or more of positions 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106; wherein a. when a substitution occurs at position 94, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr; b. when a substitution occurs at position 95, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; c. when a substitution occurs at position 96, the substituting amino acid is Asp, Gln, Glu or Ser; but when position 96 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; d. when a substitution occurs at position 97, the substituting amino acid is Asp or Glu; but when position 97 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; e. when a substitution occurs at position 98, the substituting amino acid is Asp or Glu; but when position 98 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; f. when a substitution occurs at position 99, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 99 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; g. when a substitution occurs at position 100, the substituting amino acid is Asp or Glu; but when position 100 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; h. when a substitution occurs at position 101, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 101 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103,155, 165, 188, and 216; i. when a substitution occurs at position 102, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 102 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; j. when a substitution occurs at position 103, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; k. when a substitution occurs at position 104, the substituting amino acid is Asp or Glu; l. when a substitution occurs at position 105, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; and m. when a substitution occurs at position 106, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 106 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; C. when a substitution occurs in the third loop region, the substitution occurs at one or more of positions 125, 126, 127, 128, 129, 130, 131 or 132; wherein a. when a substitution occurs at position 125, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; but when position 125 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; b. when a substitution occurs at position 126, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 126 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; c. when a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 127 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; d. when a substitution occurs at position 128, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; but when position 128 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; e. when a substitution occurs at position 129, the substituting amino acid is Asp or Glu; f. when a substitution occurs at position 130, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; g. when a substitution occurs at position 131, the substituting amino acid is Asp or Glu; and h. when a substitution occurs at position 132, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; D. when a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 or 166; wherein a. when a substitution occurs at position 153, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 153 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; b. when a substitution occurs at position 154, the substituting amino acid is Asp, Gln, Glu or Ser; c. when a substitution occurs at position 155, the substituting amino acid is Asp or Glu; d. when a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 156 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; e. when a substitution occurs at position 157, the substituting amino acid is Asp or Glu; but when position 157 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; f. when a substitution occurs at position 158, the substituting amino acid is Asp or Glu; but when position 158 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; g. when a substitution occurs at position 159, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 159 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; h. when a substitution occurs at position 160, the substituting amino acid is Asp or Glu; but when position 160 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; i. when a substitution occurs at position 161, the substituting amino acid is Asn, Asp, Glu or Ser; but when position 161 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; j. when a substitution occurs at position 162, the substituting amino acid is Asp, Gln, Glu or Ser; but when position 162 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; k. when a substitution occurs at position 163, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 163 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; l. when a substitution occurs at position 164, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 164 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; m. when a substitution occurs at position 165, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and n. when a substitution occurs at position 166, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 166 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; E. when a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 186, 187, 188, 189 or 190; wherein a. when a substitution occurs at position 186, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; b. when a substitution occurs at position 187, the substituting amino acid is Asp or Glu; c. when a substitution occurs at position 188, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val; d. when a substitution occurs at position 189, the substituting amino acid is Asp or Glu; and e. when a substitution occurs at position 190, the substituting amino acid is Asp or Glu; and F. when a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218 or 219; wherein a. when a substitution occurs at position 199, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; b. when a substitution occurs at position 200, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; c. when a substitution occurs at position 201, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 202, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr; e. when a substitution occurs at position 203, the substituting amino acid is Asp or Glu; f. when a substitution occurs at position 204, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser, or Thr; g. when a substitution occurs at position 205, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; h. when a substitution occurs at position 206, the substituting amino acid is Asp or Glu; i. when a substitution occurs at position 207, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; j. when a substitution occurs at position 208, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr or Val; k. when a substitution occurs at position 209, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; l. when a substitution occurs at position 210, the substituting amino acid is Asp or Glu; m. when a substitution occurs at position 211, the substituting amino acid is Asp, Gln, Glu or Ser; n. when a substitution occurs at position 212, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 212 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; o. when a substitution occurs at position 213, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 213 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; p. when a substitution occurs at position 214, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 214 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; q. when a substitution occurs at position 215, the substituting amino acid is Asp or Glu; r. when a substitution occurs at position 216, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; s. when a substitution occurs at position 217, the substituting amino acid is Asp, Gln, Glu or Ser; but wherein when the substituting amino acid at position 217 is Ser, the variant is not a double mutation variant having a substitution of Ser at a position selected from the group consisting of 130 and 165; t. when a substitution occurs at position 218, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and u. when a substitution occurs at position 219, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; whereby the Subtilisin DY variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type Subtilisin DY and wherein glutamate and aspartate substitutions at both subtilisin DY positions 207 and 213, or substitutions at either of 207 and 213 combined with a serine substitution at position 217, are combined with at least one further substitution at a corresponding position selected from subtilisin DY positions 59, 60, 64, 65, 104, 105, 129, 131, 132, 187, 189, 190, 199 to 202, 204, 206, 209 to 211, 215 and 219 and wherein glutamate and aspartate substitutions at both subtilisin DY positions 61 and 165 are combined with at least one further substitution at a corresponding position selected from subtilisin DY positions 59, 60, 64, 65, 104, 105, 129, 131, 132, 187, 189, 190, 199 to 202, 204, 206, 209 to 211, 215 and
 219. 9. The Subtilisin DY variant of claim 8, wherein two or more substitutions occur in the first loop region.
 10. The Subtilisin DY variant of claim 8, wherein two or more substitutions occur in the second loop region.
 11. The Subtilisin DY variant of claim 8, wherein two or more substitutions occur in the third loop region.
 12. The Subtilisin DY variant of claim 8, wherein two or more substitutions occur in the fourth loop region.
 13. The Subtilisin DY variant of claim 8, wherein two or more substitutions occur in the fifth loop region.
 14. The Subtilisin DY variant of claim 8, wherein two or more substitutions occur in the sixth loop region.
 15. A Subtilisin DY variant having a modified amino acid sequence of the Subtilisin DY wild-type amino acid sequence as set forth in SEQ ID NO:1, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region, a fifth loop region and a sixth loop region; wherein the modified amino acid sequence comprises a substitution at two or more positions in one or more of the loop regions; wherein A. when a substitution occurs in the first loop region, the substitution occurs at one of positions 60, 64 or 65; wherein a. when a substitution occurs at position 60, the substituting amino acid is Asn, Gln, Pro or Ser; b. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and c. when a substitution occurs at position 65, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; B. when a substitution occurs in the second loop region, the substitution occurs at one or more of positions 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106; wherein a. when a substitution occurs at position 94, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr; b. when a substitution occurs at position 95, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; c. when a substitution occurs at position 96, the substituting amino acid is Asp, Gln, Glu or Ser; but when position 96 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; d. when a substitution occurs at position 97, the substituting amino acid is Asp or Glu; but when position 97 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; e. when a substitution occurs at position 98, the substituting amino acid is Asp or Glu; but when position 98 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; f. when a substitution occurs at position 99, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 99 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; g. when a substitution occurs at position 100, the substituting amino acid is Asp or Glu; but when position 100 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; h. when a substitution occurs at position 101, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 101 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; i. when a substitution occurs at position 102, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 102 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; j. when a substitution occurs at position 103, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; k. when a substitution occurs at position 104, the substituting amino acid is Asp or Glu; l. when a substitution occurs at position 105, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; and m. when a substitution occurs at position 106, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 106 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; C. when a substitution occurs in the third loop region, the substitution occurs at one or more of positions 125, 126, 127, 128, 129, 130, 131 or 132; wherein a. when a substitution occurs at position 125, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; but when position 125 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; b. when a substitution occurs at position 126, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 126 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; c. when a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 127 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; d. when a substitution occurs at position 128, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; but when position 128 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; e. when a substitution occurs at position 129, the substituting amino acid is Asp or Glu; f. when a substitution occurs at position 130, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; g. when a substitution occurs at position 131, the substituting amino acid is Asp or Glu; and h. when a substitution occurs at position 132, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; D. when a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 or 166; wherein a. when a substitution occurs at position 153, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 153 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; b. when a substitution occurs at position 154, the substituting amino acid is Asp, Gln, Glu or Ser; c. when a substitution occurs at position 155, the substituting amino acid is Asp or Glu; d. when a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 156 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; e. when a substitution occurs at position 157, the substituting amino acid is Asp or Glu; but when position 157 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; f. when a substitution occurs at position 158, the substituting amino acid is Asp or Glu; but when position 158 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; g. when a substitution occurs at position 159, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 159 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; h. when a substitution occurs at position 160, the substituting amino acid is Asp or Glu; but when position 160 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; i. when a substitution occurs at position 161, the substituting amino acid is Asn, Asp, Glu or Ser; but when position 161 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; j. when a substitution occurs at position 162, the substituting amino acid is Asp, Gln, Glu or Ser; but when position 162 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; k. when a substitution occurs at position 163, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 163 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; l. when a substitution occurs at position 164, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 164 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; m. when a substitution occurs at position 165, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and n. when a substitution occurs at position 166, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 166 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; E. when a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 186, 187, 188, 189 or 190; wherein a. when a substitution occurs at position 186, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; b. when a substitution occurs at position 187, the substituting amino acid is Asp or Glu; c. when a substitution occurs at position 188, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val; d. when a substitution occurs at position 189, the substituting amino acid is Asp or Glu; and e. when a substitution occurs at position 190, the substituting amino acid is Asp or Glu; and F. when a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218 or 219; wherein a. when a substitution occurs at position 199, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; b. when a substitution occurs at position 200, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; c. when a substitution occurs at position 201, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 202, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr; e. when a substitution occurs at position 203, the substituting amino acid is Asp or Glu; f. when a substitution occurs at position 204, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser, or Thr; g. when a substitution occurs at position 205, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; h. when a substitution occurs at position 206, the substituting amino acid is Asp or Glu; i. when a substitution occurs at position 207, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; j. when a substitution occurs at position 208, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr or Val; k. when a substitution occurs at position 209, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; l. when a substitution occurs at position 210, the substituting amino acid is Asp or Glu; m. when a substitution occurs at position 211, the substituting amino acid is Asp, Gln, Glu or Ser; n. when a substitution occurs at position 212, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 212 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; o. when a substitution occurs at position 213, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 213 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; p. when a substitution occurs at position 214, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 214 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; q. when a substitution occurs at position 215, the substituting amino acid is Asp or Glu; r. when a substitution occurs at position 216, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; s. when a substitution occurs at position 217, the substituting amino acid is Asp, Gln, Glu or Ser; t. when a substitution occurs at position 218, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and u. when a substitution occurs at position 219, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; whereby the Subtilisin DY variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type Subtilisin DY and wherein glutamate and aspartate substitutions at both subtilisin DY positions 207 and 213, or substitutions at either of 207 and 213 combined with a serine substitution at position 217, are combined with at least one further substitution at a corresponding position selected from subtilisin DY positions 59, 60, 64, 65, 104, 105, 129, 131, 132, 187, 189, 190, 199 to 202, 204, 206, 209 to 211, 215 and 219 and wherein glutamate and aspartate substitutions at both subtilisin DY positions 61 and 165 are combined with at least one further substitution at a corresponding position selected from subtilisin DY positions 59, 60, 64, 65, 104, 105, 129, 131, 132, 187, 189, 190, 199 to 202, 204, 206, 209 to 211, 215 and
 219. 16. The Subtilisin DY variant of claim 15, wherein two or more substitutions occur in the first loop region.
 17. The Subtilisin DY variant of claim 15, wherein two or more substitutions occur in the second loop region.
 18. The Subtilisin DY variant of claim 15, wherein two or more substitutions occur in the third loop region.
 19. The Subtilisin DY variant of claim 15, wherein two or more substitutions occur in the fourth loop region.
 20. The Subtilisin DY variant of claim 15, wherein two or more substitutions occur in the fifth loop region.
 21. The Subtilisin DY variant of claim 15, wherein two or more substitutions occur in the sixth loop region.
 22. A Subtilisin DY variant having a modified amino acid sequence of the Subtilisin DY wild-type amino acid sequence set forth in SEQ ID NO:1, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region, a fifth loop region and a sixth loop region; wherein the modified amino acid sequence comprises a substitution at two or more positions in one or more of the loop regions; wherein A. when a substitution occurs in the first loop region, the substitution occurs at one or more of positions 58, 59, 60, 61, 62, 64 or 65; wherein a. when a substitution occurs at position 58, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; b. when a substitution occurs at position 59, the substituting amino acid is Glu; c. when a substitution occurs at position 60, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 61, the substituting amino acid is Asp, Gln, Glu or Ser; e. when a substitution occurs at position 62, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; f. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and g. when a substitution occurs at position 65, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; B. when a substitution occurs in the second loop region, the substitution occurs at one or more of positions 94, 95, 96, 99, 101, 102, 103, 105 or 106; wherein a. when a substitution occurs at position 94, the substituting amino acid is Ala, Cys, Gln, Gly, His, Met, Pro or Ser; b. when a substitution occurs at position 95, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; c. when a substitution occurs at position 96, the substituting amino acid is Gln or Ser; but when position 96 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; d. when a substitution occurs at position 99, the substituting amino acid is Asn, Gln, Pro or Ser; but when position 99 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; e. when a substitution occurs at position 101, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 101 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; f. when a substitution occurs at position 102, the substituting amino acid is Asn, Gln, Gly, Pro or Ser; but when position 102 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; g. when a substitution occurs at position 103, the substituting amino acid is Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Met, Pro or Ser; h. when a substitution occurs at position 105, the substituting amino acid is Asn, Gln, Gly, His, Pro, Ser or Thr; and i. when a substitution occurs at position 106, the substituting amino acid is Ala, Cys, Gln, Gly, His, Met, Pro, Ser or Val; but when position 106 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; C. when a substitution occurs in the third loop region, the substitution occurs at one or more of positions 125, 126, 127, 128, 129, 130, 131 or 132; wherein a. when a substitution occurs at position 125, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; but when position 125 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; b. when a substitution occurs at position 126, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 126 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; c. when a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 127 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; d. when a substitution occurs at position 128, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; but when position 128 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; e. when a substitution occurs at position 129, the substituting amino acid is Asp or Glu; f. when a substitution occurs at position 130, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; g. when a substitution occurs at position 131, the substituting amino acid is Asp or Glu; and h. when a substitution occurs at position 132, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; D. when a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 or 166; wherein a. when a substitution occurs at position 153, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 153 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; b. when a substitution occurs at position 154, the substituting amino acid is Asp, Gln, Glu or Ser; c. when a substitution occurs at position 155, the substituting amino acid is Asp or Glu; d. when a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 156 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; e. when a substitution occurs at position 157, the substituting amino acid is Asp or Glu; but when position 157 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; f. when a substitution occurs at position 158, the substituting amino acid is Asp or Glu; but when position 158 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; g. when a substitution occurs at position 159, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 159 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; h. when a substitution occurs at position 160, the substituting amino acid is Asp or Glu; but when position 160 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; i. when a substitution occurs at position 161, the substituting amino acid is Asn, Asp, Glu or Ser; but when position 161 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; j. when a substitution occurs at position 162, the substituting amino acid is Asp, Gln, Glu or Ser; but when position 162 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; k. when a substitution occurs at position 163, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 163 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; l. when a substitution occurs at position 164, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 164 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; m. when a substitution occurs at position 165, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and n. when a substitution occurs at position 166, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 166 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; E. when a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 186, 187, 188, 189 or 190; wherein a. when a substitution occurs at position 186, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; b. when a substitution occurs at position 187, the substituting amino acid is Asp or Glu; c. when a substitution occurs at position 188, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val; d. when a substitution occurs at position 189, the substituting amino acid is Asp or Glu; and e. when a substitution occurs at position 190, the substituting amino acid is Asp or Glu; and F. when a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218 or 219; wherein a. when a substitution occurs at position 199, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; b. when a substitution occurs at position 200, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; c. when a substitution occurs at position 201, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 202, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr; e. when a substitution occurs at position 203, the substituting amino acid is Asp or Glu; f. when a substitution occurs at position 204, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser, or Thr; g. when a substitution occurs at position 205, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; h. when a substitution occurs at position 206, the substituting amino acid is Asp or Glu; i. when a substitution occurs at position 207, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; j. when a substitution occurs at position 208, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr or Val; k. when a substitution occurs at position 209, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; l. when a substitution occurs at position 210, the substituting amino acid is Asp or Glu; m. when a substitution occurs at position 211, the substituting amino acid is Asp, Gln, Glu or Ser; n. when a substitution occurs at position 212, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 212 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; o. when a substitution occurs at position 213, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 213 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; p. when a substitution occurs at position 214, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 214 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; q. when a substitution occurs at position 215, the substituting amino acid is Asp or Glu; r. when a substitution occurs at position 216, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; s. when a substitution occurs at position 217, the substituting amino acid is Asp, Gln, Glu or Ser; t. when a substitution occurs at position 218, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and u. when a substitution occurs at position 219, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; whereby the Subtilisin DY variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type Subtilisin DY and wherein glutamate and aspartate substitutions at both subtilisin DY positions 207 and 213, or substitutions at either of 207 and 213 combined with a serine substitution at position 217, are combined with at least one further substitution at a corresponding position selected from subtilisin DY positions 59, 60, 64, 65, 104, 105, 129, 131, 132, 187, 189, 190, 199 to 202, 204, 206, 209 to 211, 215 and 219 and wherein glutamate and aspartate substitutions at both subtilisin DY positions 61 and 165 are combined with at least one further substitution at a corresponding position selected from subtilisin DY positions 59, 60, 64, 65, 104, 105, 129, 131, 132, 187, 189, 190, 199 to 202, 204, 206, 209 to 211, 215 and
 219. 23. The Subtilisin DY variant of claim 22, wherein two or more substitutions occur in the first loop region.
 24. The Subtilisin DY variant of claim 22, wherein two or more substitutions occur in the second loop region.
 25. The Subtilisin DY variant of claim 22, wherein two or more substitutions occur in the third loop region.
 26. The Subtilisin DY variant of claim 22, wherein two or more substitutions occur in the fourth loop region.
 27. The Subtilisin DY variant of claim 22, wherein two or more substitutions occur in the fifth loop region.
 28. The Subtilisin DY variant of claim 12, wherein two or more substitutions occur in the sixth loop region.
 29. A Subtilisin DY variant having a modified amino acid sequence of the Subtilisin DY wild-type amino acid sequence as set forth in SEQ ID NO:1, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region, a fifth loop region and a sixth loop region; wherein the modified amino acid sequence comprises a substitution at two or more positions in one or more of the loop regions; wherein A. when a substitution occurs in the first loop region, the substitution occurs at one or more of positions 58, 59, 60, 61, 62, 64 or 65; wherein a. when a substitution occurs at position 58, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; b. when a substitution occurs at position 59, the substituting amino acid is Glu; c. when a substitution occurs at position 60, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 61, the substituting amino acid is Asp, Gln, Glu or Ser; e. when a substitution occurs at position 62, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; f. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and g. when a substitution occurs at position 65, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; B. when a substitution occurs in the second loop region, the substitution occurs at one or more of positions 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106; wherein a. when a substitution occurs at position 94, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr; b. when a substitution occurs at position 95, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; c. when a substitution occurs at position 96, the substituting amino acid is Asp, Gln, Glu or Ser; but when position 96 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; d. when a substitution occurs at position 97, the substituting amino acid is Asp or Glu; but when position 97 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; e. when a substitution occurs at position 98, the substituting amino acid is Asp or Glu; but when position 98 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; f. when a substitution occurs at position 99, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 99 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; g. when a substitution occurs at position 100, the substituting amino acid is Asp or Glu; but when position 100 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; h. when a substitution occurs at position 101, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 101 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; i. when a substitution occurs at position 102, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 102 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; j. when a substitution occurs at position 103, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; k. when a substitution occurs at position 104, the substituting amino acid is Asp or Glu; l. when a substitution occurs at position 105, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; and m. when a substitution occurs at position 106, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 106 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; C. when a substitution occurs in the third loop region, the substitution occurs at one or more of positions 125, 126, 128, 131 or 132; wherein a. when a substitution occurs at position 125, the substituting amino acid is Ala, Asn, Cys, Gln, Gly, His, Met, Pro, Ser, Thr or Val; but when position 125 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; b. when a substitution occurs at position 126, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 126 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; c. when a substitution occurs at position 128, the substituting amino acid is Asn, Gln or Ser; but when position 128 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; d. when a substitution occurs at position 131, the substituting amino acid is Asp or Glu; and e. when a substitution occurs at position 132, the substituting amino acid is Asn, Gln, Gly, Pro or Ser; D. when a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 or 166; wherein a. when a substitution occurs at position 153, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 153 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; b. when a substitution occurs at position 154, the substituting amino acid is Asp, Gln, Glu or Ser; c. when a substitution occurs at position 155, the substituting amino acid is Asp or Glu; d. when a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 156 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; e. when a substitution occurs at position 157, the substituting amino acid is Asp or Glu; but when position 157 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; f. when a substitution occurs at position 158, the substituting amino acid is Asp or Glu; but when position 158 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; g. when a substitution occurs at position 159, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 159 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; h. when a substitution occurs at position 160, the substituting amino acid is Asp or Glu; but when position 160 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; i. when a substitution occurs at position 161, the substituting amino acid is Asn, Asp, Glu or Ser; but when position 161 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; j. when a substitution occurs at position 162, the substituting amino acid is Asp, Gln, Glu or Ser; but when position 162 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; k. when a substitution occurs at position 163, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 163 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; l. when a substitution occurs at position 164, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 164 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; m. when a substitution occurs at position 165, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and n. when a substitution occurs at position 166, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 166 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; E. when a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 186, 187, 188, 189 or 190; wherein a. when a substitution occurs at position 186, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; b. when a substitution occurs at position 187, the substituting amino acid is Asp or Glu; c. when a substitution occurs at position 188, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val; d. when a substitution occurs at position 189, the substituting amino acid is Asp or Glu; and e. when a substitution occurs at position 190, the substituting amino acid is Asp or Glu; and F. when a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218 or 219; wherein a. when a substitution occurs at position 199, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; b. when a substitution occurs at position 200, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; c. when a substitution occurs at position 201, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 202, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr; e. when a substitution occurs at position 203, the substituting amino acid is Asp or Glu; f. when a substitution occurs at position 204, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser, or Thr; g. when a substitution occurs at position 205, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; h. when a substitution occurs at position 206, the substituting amino acid is Asp or Glu; i. when a substitution occurs at position 207, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; j. when a substitution occurs at position 208, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr or Val; k. when a substitution occurs at position 209, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; l. when a substitution occurs at position 210, the substituting amino acid is Asp or Glu; m. when a substitution occurs at position 211, the substituting amino acid is Asp, Gln, Glu or Ser; n. when a substitution occurs at position 212, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 212 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; o. when a substitution occurs at position 213, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 213 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; p. when a substitution occurs at position 214, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 214 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; q. when a substitution occurs at position 215, the substituting amino acid is Asp or Glu; r. when a substitution occurs at position 216, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; s. when a substitution occurs at position 217, the substituting amino acid is Asp, Gln, Glu or Ser; t. when a substitution occurs at position 218, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and u. when a substitution occurs at position 219, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; whereby the Subtilisin DY variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type Subtilisin DY and wherein glutamate and aspartate substitutions at both subtilisin DY positions 207 and 213, or substitutions at either of 207 and 213 combined with a serine substitution at position 217, are combined with at least one further substitution at a corresponding position selected from subtilisin DY positions 59, 60, 64, 65, 104, 105, 129, 131, 132, 187, 189, 190, 199 to 202, 204, 206, 209 to 211, 215 and 219 and wherein glutamate and aspartate substitutions at both subtilisin DY positions 61 and 165 are combined with at least one further substitution at a corresponding position selected from subtilisin DY positions 59, 60, 64, 65, 104, 105, 129, 131, 132, 187, 189, 190, 199 to 202, 204, 206, 209 to 211, 215 and
 219. 30. The Subtilisin DY variant of claim 29, wherein two or more substitutions occur in the first loop region.
 31. The Subtilisin DY variant of claim 29, wherein two or more substitutions occur in the second loop region.
 32. The Subtilisin DY variant of claim 29, wherein two or more substitutions occur in the third loop region.
 33. The Subtilisin DY variant of claim 29, wherein two or more substitutions occur in the fourth loop region.
 34. The Subtilisin DY variant of claim 29, wherein two or more substitutions occur in the fifth loop region.
 35. The Subtilisin DY variant of claim 29, wherein two or more substitutions occur in the sixth loop region.
 36. A Subtilisin DY variant having a modified amino acid sequence of the Subtilisin DY wild-type amino acid sequence as set forth in SEQ ID NO:1, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region, a fifth loop region and a sixth loop region; wherein the modified amino acid sequence comprises a substitution at two or more positions in one or more of the loop regions; wherein A. when a substitution occurs in the first loop region, the substitution occurs at one or more of positions 58, 59, 60, 61, 62, 64 or 65; wherein a. when a substitution occurs at position 58, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; b. when a substitution occurs at position 59, the substituting amino acid is Glu; c. when a substitution occurs at position 60, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 61, the substituting amino acid is Asp, Gln, Glu or Ser; e. when a substitution occurs at position 62, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; f. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and g. when a substitution occurs at position 65, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; B. when a substitution occurs in the second loop region, the substitution occurs at one or more of positions 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106; wherein a. when a substitution occurs at position 94, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr; b. when a substitution occurs at position 95, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; c. when a substitution occurs at position 96, the substituting amino acid is Asp, Gln, Glu or Ser; but when position 96 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; d. when a substitution occurs at position 97, the substituting amino acid is Asp or Glu; but when position 97 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; e. when a substitution occurs at position 98, the substituting amino acid is Asp or Glu; but when position 98 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; f. when a substitution occurs at position 99, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 99 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; g. when a substitution occurs at position 100, the substituting amino acid is Asp or Glu; but when position 100 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; h. when a substitution occurs at position 101, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 101 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; i. when a substitution occurs at position 102, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 102 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; j. when a substitution occurs at position 103, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; k. when a substitution occurs at position 104, the substituting amino acid is Asp or Glu; l. when a substitution occurs at position 105, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; and m. when a substitution occurs at position 106, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 106 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; C. when a substitution occurs in the third loop region, the substitution occurs at one or more of positions 125, 126, 127, 128, 129, 130, 131 or 132; wherein a. when a substitution occurs at position 125, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; but when position 125 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; b. when a substitution occurs at position 126, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 126 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; c. when a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 127 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; d. when a substitution occurs at position 128, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; but when position 128 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; e. when a substitution occurs at position 129, the substituting amino acid is Asp or Glu; f. when a substitution occurs at position 130, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; g. when a substitution occurs at position 131, the substituting amino acid is Asp or Glu; and h. when a substitution occurs at position 132, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; D. when a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 154, 156 or 166; wherein a. when a substitution occurs at position 154, the substituting amino acid is Gln or Ser; b. when a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 156 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; and c. when a substitution occurs at position 166, the substituting amino acid is Ala, Gly, His, Ile, Leu, Met or Pro; but when position 166 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; E. when a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 186, 187, 188, 189 or 190; wherein a. when a substitution occurs at position 186, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; b. when a substitution occurs at position 187, the substituting amino acid is Asp or Glu; c. when a substitution occurs at position 188, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val; d. when a substitution occurs at position 189, the substituting amino acid is Asp or Glu; and e. when a substitution occurs at position 190, the substituting amino acid is Asp or Glu; and F. when a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218 or 219; wherein a. when a substitution occurs at position 199, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; b. when a substitution occurs at position 200, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; c. when a substitution occurs at position 201, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 202, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr; e. when a substitution occurs at position 203, the substituting amino acid is Asp or Glu; f. when a substitution occurs at position 204, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser, or Thr; g. when a substitution occurs at position 205, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; h. when a substitution occurs at position 206, the substituting amino acid is Asp or Glu; i. when a substitution occurs at position 207, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; j. when a substitution occurs at position 208, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr or Val; k. when a substitution occurs at position 209, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; l. when a substitution occurs at position 210, the substituting amino acid is Asp or Glu; m. when a substitution occurs at position 21 1, the substituting amino acid is Asp, Gln, Glu or Ser; n. when a substitution occurs at position 212, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 212 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; o. when a substitution occurs at position 213, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 213 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; p. when a substitution occurs at position 214, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 214 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 103, 188, and 216; q. when a substitution occurs at position 215, the substituting amino acid is Asp or Glu; r. when a substitution occurs at position 216, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; s. when a substitution occurs at position 217, the substituting amino acid is Asp, Gln, Glu or Ser; t. when a substitution occurs at position 218, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and u. when a substitution occurs at position 219, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; whereby the Subtilisin DY variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type Subtilisin DY and wherein glutamate and aspartate substitutions at both subtilisin DY positions 207 and 213, or substitutions at either of 207 and 213 combined with a serine substitution at position 217, are combined with at least one further substitution at a corresponding position selected from subtilisin DY positions 59, 60, 64, 65, 104, 105, 129, 131, 132, 187, 189, 190, 199 to 202, 204, 206, 209 to 211, 215 and 219 and wherein glutamate and aspartate substitutions at both subtilisin DY positions 61 and 165 are combined with at least one further substitution at a corresponding position selected from subtilisin DY positions 59, 60, 64, 65, 104, 105, 129, 131, 132, 187, 189, 190, 199 to 202, 204, 206, 209 to 211, 215 and
 219. 37. The Subtilisin DY variant of claim 36, wherein two or more substitutions occur in the first loop region.
 38. The Subtilisin DY variant of claim 36, wherein two or more substitutions occur in the second loop region.
 39. The Subtilisin DY variant of claim 36, wherein two or more substitutions occur in the third loop region.
 40. The Subtilisin DY variant of claim 36, wherein two or more substitutions occur in the fourth loop region.
 41. The Subtilisin DY variant of claim 36, wherein two or more substitutions occur in the fifth loop region.
 42. The Subtilisin DY variant of claim 36, wherein two or more substitutions occur in the sixth loop region.
 43. A Subtilisin DY variant having a modified amino acid sequence of the Subtilisin DY wild-type amino acid sequence as set forth in SEQ ID NO:1, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region, a fifth loop region and a sixth loop region; wherein the modified amino acid sequence comprises a substitution at two or more positions in one or more of the loop regions; wherein A. when a substitution occurs in the first loop region, the substitution occurs at one or more of positions 58, 59, 60, 61, 62, 64 or 65; wherein a. when a substitution occurs at position 58, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; b. when a substitution occurs at position 59, the substituting amino acid is Glu; c. when a substitution occurs at position 60, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 61, the substituting amino acid is Asp, Gln, Glu or Ser; e. when a substitution occurs at position 62, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; f. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and g. when a substitution occurs at position 65, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; B. when a substitution occurs in the second loop region, the substitution occurs at one or more of positions 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106; wherein a. when a substitution occurs at position 94, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr; b. when a substitution occurs at position 95, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; c. when a substitution occurs at position 96, the substituting amino acid is Asp, Gln, Glu or Ser; but when position 96 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; d. when a substitution occurs at position 97, the substituting amino acid is Asp or Glu; but when position 97 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; e. when a substitution occurs at position 98, the substituting amino acid is Asp or Glu; but when position 98 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; f. when a substitution occurs at position 99, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 99 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; g. when a substitution occurs at position 100, the substituting amino acid is Asp or Glu; but when position 100 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; h. when a substitution occurs at position 101, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 101 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; i. when a substitution occurs at position 102, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 102 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; j. when a substitution occurs at position 103, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; k. when a substitution occurs at position 104, the substituting amino acid is Asp or Glu; l. when a substitution occurs at position 105, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; and m. when a substitution occurs at position 106, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 106 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; C. when a substitution occurs in the third loop region, the substitution occurs at one or more of positions 125, 126, 127, 128, 129, 130, 131 or 132; wherein a. when a substitution occurs at position 125, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; but when position 125 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; b. when a substitution occurs at position 126, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 126 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; c. when a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 127 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; d. when a substitution occurs at position 128, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; but when position 128 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; e. when a substitution occurs at position 129, the substituting amino acid is Asp or Glu; f. when a substitution occurs at position 130, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; g. when a substitution occurs at position 131, the substituting amino acid is Asp or Glu; and h. when a substitution occurs at position 132, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; D. when a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 or 166; wherein a. when a substitution occurs at position 153, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 153 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; b. when a substitution occurs at position 154, the substituting amino acid is Asp, Gln, Glu or Ser; c. when a substitution occurs at position 155, the substituting amino acid is Asp or Glu; d. when a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 156 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; e. when a substitution occurs at position 157, the substituting amino acid is Asp or Glu; but when position 157 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; f. when a substitution occurs at position 158, the substituting amino acid is Asp or Glu; but when position 158 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; g. when a substitution occurs at position 159, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 159 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; h. when a substitution occurs at position 160, the substituting amino acid is Asp or Glu; but when position 160 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; i. when a substitution occurs at position 161, the substituting amino acid is Asn, Asp, Glu or Ser; but when position 161 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; j. when a substitution occurs at position 162, the substituting amino acid is Asp, Gln, Glu or Ser; but when position 162 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; k. when a substitution occurs at position 163, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 163 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; l. when a substitution occurs at position 164, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 164 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; m. when a substitution occurs at position 165, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and n. when a substitution occurs at position 166, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 166 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; E. when a substitution occurs in the fifth loop region, the substitution occurs at position 188; wherein the substituting amino acid is Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val; and F. when a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218 or 219; wherein a. when a substitution occurs at position 199, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; b. when a substitution occurs at position 200, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; c. when a substitution occurs at position 201, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 202, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr; e. when a substitution occurs at position 203, the substituting amino acid is Asp or Glu; f. when a substitution occurs at position 204, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser, or Thr; g. when a substitution occurs at position 205, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; h. when a substitution occurs at position 206, the substituting amino acid is Asp or Glu; i. when a substitution occurs at position 207, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; j. when a substitution occurs at position 208, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr or Val; k. when a substitution occurs at position 209, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; l. when a substitution occurs at position 210, the substituting amino acid is Asp or Glu; m. when a substitution occurs at position 211, the substituting amino acid is Asp, Gln, Glu or Ser; n. when a substitution occurs at position 212, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 212 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; o. when a substitution occurs at position 213, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 213 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; p. when a substitution occurs at position 214, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 214 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; q. when a substitution occurs at position 215, the substituting amino acid is Asp or Glu; r. when a substitution occurs at position 216, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; s. when a substitution occurs at position 217, the substituting amino acid is Asp, Gln, Glu or Ser; t. when a substitution occurs at position 218, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and u. when a substitution occurs at position 219, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; whereby the Subtilisin DY variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type Subtilisin DY and wherein glutamate and aspartate substitutions at both subtilisin DY positions 207 and 213, or substitutions at either of 207 and 213 combined with a serine substitution at position 217, are combined with at least one further substitution at a corresponding position selected from subtilisin DY positions 59, 60, 64, 65, 104, 105, 129, 131, 132, 187, 189, 190, 199 to 202, 204, 206, 209 to 211, 215 and 219 and wherein glutamate and aspartate substitutions at both subtilisin DY positions 61 and 165 are combined with at least one further substitution at a corresponding position selected from subtilisin DY positions 59, 60, 64, 65, 104, 105, 129, 131, 132, 187, 189, 190, 199 to 202, 204, 206, 209 to 211, 215 and
 219. 44. The Subtilisin DY variant of claim 43, wherein two or more substitutions occur in the first loop region.
 45. The Subtilisin DY variant of claim 43, wherein two or more substitutions occur in the second loop region.
 46. The Subtilisin DY variant of claim 43, wherein two or more substitutions occur in the third loop region.
 47. The Subtilisin DY variant of claim 43, wherein two or more substitutions occur in the fourth loop region.
 48. The Subtilisin DY variant of claim 43, wherein one substitution occurs in the fifth loop region.
 49. The Subtilisin DY variant of claim 43, wherein two or more substitutions occur in the sixth loop region.
 50. A Subtilisin DY variant having a modified amino acid sequence of the Subtilisin DY wild-type amino acid sequence as set forth in SEQ ID NO:1, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region, a fifth loop region and a sixth loop region; wherein the modified amino acid sequence comprises a substitution at two or more positions in one or more of the loop regions; wherein A. when a substitution occurs in the first loop region, the substitution occurs at one or more of positions 58, 59, 60, 61, 62, 64 or 65; wherein a. when a substitution occurs at position 58, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; b. when a substitution occurs at position 59, the substituting amino acid is Glu; c. when a substitution occurs at position 60, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 61, the substituting amino acid is Asp, Gln, Glu or Ser; e. when a substitution occurs at position 62, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; f. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and g. when a substitution occurs at position 65, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; B. when a substitution occurs in the second loop region, the substitution occurs at one or more of positions 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106; wherein a. when a substitution occurs at position 94, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr; b. when a substitution occurs at position 95, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; c. when a substitution occurs at position 96, the substituting amino acid is Asp, Gln, Glu or Ser; but when position 96 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; d. when a substitution occurs at position 97, the substituting amino acid is Asp or Glu; but when position 97 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; e. when a substitution occurs at position 98, the substituting amino acid is Asp or Glu; but when position 98 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; f. when a substitution occurs at position 99, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 99 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; g. when a substitution occurs at position 100, the substituting amino acid is Asp or Glu; but when position 100 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; h. when a substitution occurs at position 101, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 101 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; i. when a substitution occurs at position 102, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 102 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; j. when a substitution occurs at position 103, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; k. when a substitution occurs at position 104, the substituting amino acid is Asp or Glu; l. when a substitution occurs at position 105, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; and m. when a substitution occurs at position 106, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 106 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; C. when a substitution occurs in the third loop region, the substitution occurs at one or more of positions 125, 126, 127, 128, 129, 130, 131 or 132; wherein a. when a substitution occurs at position 125, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; but when position 125 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; b. when a substitution occurs at position 126, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 126 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; c. when a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 127 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; d. when a substitution occurs at position 128, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; but when position 128 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; e. when a substitution occurs at position 129, the substituting amino acid is Asp or Glu; f. when a substitution occurs at position 130, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; g. when a substitution occurs at position 131, the substituting amino acid is Asp or Glu; and h. when a substitution occurs at position 132, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; D. when a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 or 166; wherein a. when a substitution occurs at position 153, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 153 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; b. when a substitution occurs at position 154, the substituting amino acid is Asp, Gln, Glu or Ser; c. when a substitution occurs at position 155, the substituting amino acid is Asp or Glu; d. when a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 156 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; e. when a substitution occurs at position 157, the substituting amino acid is Asp or Glu; but when position 157 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; f. when a substitution occurs at position 158, the substituting amino acid is Asp or Glu; but when position 158 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; g. when a substitution occurs at position 159, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 159 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; h. when a substitution occurs at position 160, the substituting amino acid is Asp or Glu; but when position 160 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; i. when a substitution occurs at position 161, the substituting amino acid is Asn, Asp, Glu or Ser; but when position 161 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; j. when a substitution occurs at position 162, the substituting amino acid is Asp, Gln, Glu or Ser; but when position 162 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; k. when a substitution occurs at position 163, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; but when position 163 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; l. when a substitution occurs at position 164, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 164 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; m. when a substitution occurs at position 165, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and n. when a substitution occurs at position 166, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 166 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216; E. when a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 186, 187, 188, 189 or 190; wherein a. when a substitution occurs at position 186, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; b. when a substitution occurs at position 187, the substituting amino acid is Asp or Glu; c. when a substitution occurs at position 188, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val; d. when a substitution occurs at position 189, the substituting amino acid is Asp or Glu; and e. when a substitution occurs at position 190, the substituting amino acid is Asp or Glu; and F. when a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 199, 200, 201, 202, 204, 205, 206, 207, 208, 209, 211, 212, 213, 214, 216, or 219; wherein a. when a substitution occurs at position 199, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; b. when a substitution occurs at position 200, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser; c. when a substitution occurs at position 201, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 202, the substituting amino acid is Ala, Cys, Gln, Gly, His, Met, Pro or Ser; e. when a substitution occurs at position 204, the substituting amino acid is Ala, Cys, Gln, Gly, His, Met, Pro or Ser; f. when a substitution occurs at position 205, the substituting amino acid is Ala, Asn, Gln, Gly, His, Ile, Leu, Met, Pro or Ser; g. when a substitution occurs at position 206, the substituting amino acid is Asp or Glu; h. when a substitution occurs at position 207, the substituting amino acid is Asn, Gln, Gly, Pro or Ser; i. when a substitution occurs at position 208, the substituting amino acid is Ala, Asn, Cys, Gln, Gly, His, Ile, Met, Pro or Ser; j. when a substitution occurs at position 209, the substituting amino acid is Asn, Gln, Gly, or Ser; k. when a substitution occurs at position 211, the substituting amino acid is Gln or Ser; l. when a substitution occurs at position 212, the substituting amino acid is Asn, Gln, Gly, Pro or Ser; but when position 212 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; m. when a substitution occurs at position 213, the substituting amino acid is Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Met or Pro; but when position 213 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; n. when a substitution occurs at position 214, the substituting amino acid is Asn, Gln, Gly, Pro or Ser; but when position 214 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 103, 154, 155, 165, 188, and 216; o. when a substitution occurs at position 216, the substituting amino acid is Ala, Asn, Cys, Gln, Gly, His, Ile, Met, Pro, Ser, Thr or Val; and p. when a substitution occurs at position 219, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; whereby the Subtilisin DY variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type Subtilisin DY.
 51. The Subtilisin DY variant of claim 50, wherein two or more substitutions occur in the first loop region.
 52. The Subtilisin DY variant of claim 50, wherein two or more substitutions occur in the second loop region.
 53. The Subtilisin DY variant of claim 50, wherein two or more substitutions occur in the third loop region.
 54. The Subtilisin DY variant of claim 50, wherein two or more substitutions occur in the fourth loop region.
 55. The Subtilisin DY variant of claim 50, wherein two or more substitutions occur in the fifth loop region.
 56. The Subtilisin DY variant of claim 50, wherein two or more substitutions occur in the sixth loop region.
 57. A cleaning composition selected from the group consisting of a hard surface cleaning composition, a dishwashing composition, an oral cleaning composition, a denture cleansing composition, a contact lens cleaning composition and a fabric cleaning composition, wherein the cleaning composition comprises the Subtilisin DY variant of claim 8 and a cleaning composition carrier.
 58. The cleaning composition of claim 57, wherein the cleaning composition is a hard surface cleaning composition.
 59. The cleaning composition of claim 57, wherein the cleaning composition is a fabric cleaning composition.
 60. The fabric cleaning composition of claim 57, wherein the composition is in the form of a liquid.
 61. The fabric cleaning composition of claim 60, wherein the composition further comprises at least about 5% surfactant and at least about 5% builder, by weight of the composition.
 62. The fabric cleaning composition of claim 61 further comprising cleaning composition materials selected from the group consisting of solvents, buffers, enzymes, soil release agents, clay soil removal agents, dispersing agents, brighteners, suds supressors, fabric softeners, suds boosters, enzyme stabilizers, bleaching agents, dyes, perfumes, and mixtures thereof.
 63. The fabric cleaning composition of claim 61 further comprising at least one bleaching agent.
 64. A cleaning composition selected from the group consisting of a hard surface cleaning composition, a dishwashing composition, an oral cleaning composition, a denture cleansing composition, a contact lens cleaning composition and a fabric cleaning composition, wherein the, cleaning composition comprises the Subtilisin DY variant of claim 14 and a cleaning composition carrier.
 65. A DNA sequence encoding the Subtilisin DY variant of claim
 8. 